CONVERSION FROM AZATHIOPRINA TO MYCOPHENOLATE MOFETIL IN PEDIATRIC RENAL TRANSPLANT RECIPIENTS WITH CHRONIC REJECTION¹

Abstract

Background. 

Chronic rejection is the leading cause of graft failure.Both nonimmunological and immunological mechanisms contribute to this pathology.

Methods. 

We studied changes in kidney function, mixed lymphocyte culture, cell-mediated lympholysis, serum HLA class Ι antigens, cytotoxic antibodies, and lymphocyte population before and after 6 months of follow-up in 22 pediatric renal transplanted patients. The immunosuppressive protocol used was: cyclosporine, azathioprine, and corticosteroids. Eight patients demonstrated chronic graft rejection (by biopsy), group Ι; and eight patients had no clinical evidence of chronic and/or acute rejection, group ΙΙ. Substitution of mycophenolate mofetil (MMF) (600 mg/m² bid for azathioprine was done in patients of groups Ι and ΙΙ. Another six patients with chronic rejection, did not receive MMF, group ΙΙΙ.

Results. 

Creatinine clearance increased in group Ι (44±5 vs. 51.1±6 ml/min/1.73 m², P <0.03) but it decreased in group ΙΙΙ (30±3 vs. 25±2, P <0.01). Urinary protein excretion decreased only in group Ι (0.3±0.03 to 0.06±0.03 g/24 hr, P <0.03). During MMF therapy antidonor mixed lymphocyte culture decreased 62 and 70% (P <0.05) in group Ι and ΙΙ. Cell-mediated lympholysis against lymphocyte of the donor decreased 65% (P <0.05) in group Ι. Cell-mediated lympholysis toward control cells decreased 54% (P <0.01) in group ΙΙ. Serum HLA class Ι antigens, only decreased from 0.7±0.1 to 0.5±0.1 μl/ml, P <0.05, in group Ι. CD19⁺ decreased from 7.9±1.1 to 5.6±0.8%, P <0.05, and 7.8±1.2 to 5.5±0.9%, P <0.05, in groups Ι and ΙΙ, respectively. CD16⁺ increased from 5.7±1.1 to 8.6±1.3 (P <0.05) only in group Ι.

Conclusions. 

Our data suggest that substituting MMF for azathioprine therapy leads to an improvement in the immunosuppression and renal function in children with on-going chronic rejection.

Chronic rejection is the most common cause of graft failure after pediatric kidney transplantation (¹). Immunological and nonimmunological mechanisms contribute to this process (^{2, 3}). The histology of the chronic rejection is associated with a proliferative and obliterative arteriopathy, attributed to proliferation of smooth muscle cells and fibroblasts observed in the arterial tree of transplanted kidneys (⁴). The pathogenesis of proliferative arteriopathy is mediated by T lymphocytes and antibodies against donor antigens (^{5, 6}).

Mycophenolate mofetil (MMF) is an ester prodrug of the active immunosuppressant mycophenolic acid (⁷). It is a noncompetitive, selective, and reversible inhibitor of inosine monophosphate dehydrogenase, an enzime in the de novo synthesis of guanosine nucleotides in T and B lymphocytes (⁸). MMF inhibits: (1) the proliferation of lymphocytes and smooth muscle cells (^{9, 10}), (2) the cytotoxic T lymphocyte response to allogenic cells (⁹), and (3) the production of antibodies induced by mitogens and antigens (^{9, 11}). MMF was associated with a significantly lower proportion of patients experiencing acute rejection or treatment failure, after transplantation (¹²). Also, MMF was effective for the treatment of refractory acute rejection (¹³). Moreover, studies in animals had demonstrated that MMF may inhibit the chronic rejection process (^{14, 15}).

However, there is no information at present about its immunosuppressive activity in pediatric renal transplant patients with on-going chronic rejection. Therefore, the aim of this study was to evaluate renal function, proliferative and cytolytic responses, lymphocyte subpopulations, serum HLA class I antigens, and the occurrence of cytotoxic antibodies before and after substitution of MMF for azathioprine in pediatric transplant recipients with chronic rejection.

MATERIAL AND METHODS

All patients were treated and studied at the Pediatric Department of the Hospital Italiano of Buenos Aires. Twenty-two patients were selected out of a larger group (n=150) of pediatric renal transplantatients (Table 1). Chronic renal failure was secondary to obstructive uropathy (four patients), hemolytic uremic syndrome (six patients), focal segmental glomerulosclerosis (five patients), nephronoptisis (two patients), lupus nephritis, IgA-IgG nephropathy, Bartter syndrome, congenital nephrotic syndrome, membranous nephropaty (one patient each).

Table 1: Patients characteristic before the study protocol

Inclusion criteria were: (1) all patients had received a kidney for living related, HLA-haploidentical donor, (2) no antilymphocyte serum, or antithymocyte globulin or OKT3 monoclonal antobodies were used, and (3) no patient had acute rejection, cyclosporine toxicity, at least 6 months before the study; or renal artery stenosis.

Patients were divided in three groups according to the presence (groups Ι and ΙΙΙ) or absence (group ΙΙ) of chronic rejection. Group II included eight patients without chronic rejection who where agree to participate in the study. Group ΙΙΙ consisted of six patients with chronic rejection who refused to be treated with MMF, but they decided to stay in the protocol. Chronic rejection included: progressive deterioration of renal function and transplant glomerulopathy and/or chronic vascular changes (by core biopsy at start of study). Because this was not a controlled and randomized trial, differences exist between groups, therefore comparisons were done only within the groups and not between groups.

The immunosuppressive protocol used was a triple-drug regimen of cyclosporine (CsA), azathioprine (AZA), and corticosteroids (methylprednisone or deflazacort). The mean maintenance dosage of orally administered AZA before the start of the study was 1.8 mg/kg/day (range 1.5 to 2.0 mg/kg/day). This was replaced in groups I and II by a dosage of 600 mg/m (²) of MMF (Cellcept, Roche, Buenos Aires, Argentina).

The MMF treatment protocol was approved by the ethical committee of the Pediatric Department (Hospital Italiano). Assent was obtained from the children and donors studied and informed consent was signed by parents.

Laboratory studies were performed before and after 6 months of MMF therapy. Twenty-two living-related donor recipients combinations were investigated for their proliferative response on the basis of mixed lymphocyte culture (MLC) and cytolytic potential using cell-mediated lympholysis assay (CML). Also, patients were investigated for lymphocyte subpopulations (CD3⁺, CD4⁺, CD8⁺, CD16⁺, CD19⁺, and CD4⁺/CD8⁺ ratio), serum HLA class I antigens and ocurrence of cytotoxic antibodies in the cross-match serum. All immunosuppressive drugs were withdrawn for 12 hr before blood sample were taken from recipients for testing. Serum creatinine concentration, calculated creatinine clearance, and urinary protein excretion were determined monthy during follow-up.

Standard MLC technique were used in which the peripheral blood lymphocytes of the recipients were sensitized in vitro against donor, and against unrelated healthy subjects, and measured as a proliferative response by [H (³)] thymidine incorporation. The MLC results are expressed as relative response (RR). The RR is the ratio between the net cpm of an allogeneic combination and the net cpm in a maximally stimulated combination. The ratio is multiplied by 100 to yield a percent RR value.

The CML assay has been described previously in detail. Briefly, the receptor cells (R) were cultured with irradiated donor [RD⁺] and control cells [RC⁺] for 5 days. Then, this effector cells were cultured for 4 hr with donor (D) and control cells (C), previously stimulated with phytohemagglutinin for 3 days and marked with Cr (51) (RD⁺/D and RC⁺/C).The results were expressed as percentage of cromium liberation. Patient with percentages equal to or less than 10% were considered nonresponders, those with values from 11 to 20% were hyporesponders, and those with percentages equal to or more than 60% were considered normal responders.

Serum HLA class I antigens were measured using the serum HLA STAT class I kit (Sang Stat Medical Corp., Menlo Park, CA). The patients cytotoxic antibodies were studied with direct cross-match (anti-B cells and anti-T cells) and PRA were carried out using the microlymphocytotoxicity technique. A direct cross-match and PRA were considered positive when cells lysis was 10% or more above the background for the different cell populations. Lymphocyte subpopulations (CD3⁺, CD4⁺, CD8⁺, CD16⁺, and CD19⁺) were measured using flow cytometry (Ortho Cytoronabsolute, Ortho Diagnostic Systems, Raritan. NY).

Serum creatinine concentrations were determined using the picric acid method. Creatinine clearance was calculated using the Schwartz formula: creatinine clearance (ml/min/1.73 m2=height (cm)×0.55/serum creatinine (mg/dl). Urinary protein excretion were measured using benzethonium chloride.

Statistical analysis.

Results were analyzed by paired and unpaired Student’s t tests and linear regression as appropriate. Values are reported as mean and ranges, or as mean±SEM, P <0.05 being considered statistically significant.

RESULTS

Functional studies.

Non-MMF-treated patients with chronic rejection (group ΙΙΙ) experienced a significant decrease in renal function (P <0.01) and a non significant increase in urinary protein excretion (Table 2). In contrast in MMF-treated patients with chronic rejection (group Ι) renal function improved significantly, and in addition levels of urinary protein excretion decreased significantly (Table 2). In MMF-treated patients without chronic rejection (group ΙΙ), renal function remained stable and never demonstrated proteinuria throughout the follow-up period (15 months). The cyclosporine doses and levels did not change during the study (Table 2).

Table 2: Comparison^a of renal function, CsA doses, and levels before and during the study protocol

Immunology.

Immunological features of the non-MMF patients with chronic rejection (group ΙΙΙ) varied markedly between MMF-treated patients (groups Ι and ΙΙ). The antidonor MLC activity, is shown in Figure 1. The MMF-treated patients display a significant change (P <0.05) in MLC reactivity before and during MMF therapy; in contrast, no change were observed in non-MMF treated patients.

Figure 1: MLC study. Proliferative response before and during the study protocol.

The CML (RD⁺/D) test against the lymphocytes of the kidney donor decreased significantly (P <0.03) during MMF therapy in patients with chronic rejection (group Ι). In contrast, neither group II and group III patients changed their percentage of cell lysis (RD⁺/D) during the follow-up period (Fig. 2). The CML (RC⁺/C) test against the lymphocytes of control decreased significantly only in group II patients (P <0.01, Fig. 2).

Figure 2: CML study. Cytolytic responses against donor (D) and control (C) cells before and during the study protocol.

Percentages of CD19⁺ cells decreased significantly (P <0.05) during MMF therapy (groups Ι and ΙΙ) but, CD16⁺ cells increased (P <0.05) only in group Ι patients (Table 3). Only in MMF-treated patients with chronic rejection (group Ι) the serum HLA class Ι antigens decreased significantly (P <0.05) during treatment (Fig. 3).

Table 3: Variations in lymphocyte subpopulations (%) before and during the study protocol

Figure 3: Serum HLA class Ι antigens before and during the study protocol.

Two of 16 MMF-treated patients had cytotoxic PRA during AZA therapy. During MMF therapy only two patients had a positive antidonor B cell cross-match and none of the recipients had PRA in serum samples. In group ΙΙΙ one patient had a positive antidonor B cell cross-match and cytotoxic PRA during the follow-up period. None of patient had a positive antidonor T cell cytotoxicity cross-match during AZA or MMF treatment.

DISCUSSION

Our findings in pediatric renal transplant recipients with chronic rejection indicate that a shift from AZA to MMF results in a significant decrease in patient’s proliferative and cytolytic potencial, and in a significant decrease also in concentration of serum HLA class I antigens and CD19⁺ cells. These favorable changes in the host immune response were associated with a significant improve in graft function and urinary protein excretion. However, none of these advantages were observed in a control population with chronic rejection treated with AZA.

MMF had previously been shown effective in preventing kidney allograft acute rejection if used in combination with CsA or tacrolimus (FK 506) and prednisolone (¹²) and, in the treatment of refractory acute rejection (¹³). Reports that MMF can prevent the changes of chronic rejection of kidney allografts (¹⁵) and cardiac xenografts (¹⁴) animal models, had prompted us to investigate the use of MMF for treatment of kidney chronic rejection in pediatric patients.

Switching from AZA to MMF was not associated with the appearance of any sign of acute rejection. Moreover, only in patients with chronic rejection, serum creatinine, calculated creatinine clearance, and urinary protein excretion improved during the first 15 months of follow-up. CsA may cause dose-related reduction in renal function due to the drug-inducing preglomerular vasoconstriction (¹⁶); however, the improvement of renal function in our patients with chronic rejection were observed without changes in the CsA levels and doses.

The host immune response against vascularized organ allografts includes both cellular and humoral components. These host immune responses were investigated in our patients, for their proliferative and cytolytic responses, lymphocyte subpopulations, serum HLA class I antigens, and anti-HLA antibodies at the start of the protocol and after 6 months of follow-up. In contrast to the control group patients, in the groups treated with MMF the specific antidonor MLC reactivity decreased significantly. Also, a significant drop in CML reactivity toward the kidney donor lymphocytes and toward control cells during MMF treatment was observed in patients with and without chronic rejection, respectively. Thus, MMF therapy had greater antiproliferative effect on lymphocytes and inhibited the generation of cytotoxic T cells in our patients.

Therefore, clinically attainable concentrations of MMF may also inhibit the proliferation of human smooth muscle cells and renal instertitial cellular infiltration of cytotoxic T cells that are relevant in the process of chronic rejection (¹⁰). MMF may decrease specific CML reactivity in patients with chronic rejection by several mechanisms: (1) inhibition of inosine monophosphate dehydrogenase (⁸), (2) inhibition of the transfer of fucose and manose to glycoproteins, including adhesion molecules (^{17, 18}), (3) inhibition of interleukin-Iβ, interleukin-10, and tumor necrosis factor-α responses (¹⁹).

Total serum HLA class I antigens rise in conjunction with rejection of all allografts. This findings are consistent with the idea that serum HLA class I antigens elevations are a marker of immune activity during allogeneic transplantation. Serum HLA class I antigens may be either spontaneously shed by grafted organs or from damaged grafted cells (donor cells) or produced during rejection episodes by activate cytotoxic T cells (receptor origen) (^{20, 21}). Therefore, the reduced levels of total serum HLA class I antigens in the majority of patients with chronic rejection treated with MMF could be an expression of decreased activated T lymphocytes and adequate immunosuppressive treatment (²²). The production of anti-HLA antibodies was low and the patients, including the control group, did not form HLA antibodies during the follow-up.

Due to MMF is an inhibitor of DNA synthesis of activated T and B cells, we evaluated also, if MMF could change the percentages of lymphocytes subpopulations included T, B, and NK cells. T lymphocytes (CD3⁺, CD4⁺, and CD8⁺) did not change during MMF treatment; in contrast B lyphocytes (CD19⁺) were decreased only in patients treated with MMF. A surprising finding was the clustering toward the upper range of CD16⁺ cells only in group I patients. This finding suggests the existence of a dinamic balance between the natural killer and the cytotoxic T cell systems. The beneficial effect of natural killer cells increase, is the improvement in tumor surveillance.

Our data suggest that subtituting MMF for AZA leads to an improvement in the immunosuppression and in the renal function of the patients with chronic rejection. Therefore, these findings support the important role of immunological factors in the development of chronic graft failure. Additional and larger studies are necessary before widespread use of MMF in on going kidney chronic rejection can be advocated.