EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Here, we report our 1-year follow-up data of stable ABO-incompatible kidney transplant recipients who converted from mycophenolate mofetil plus a standard dose of a calcineurin inhibitor to everolimus plus low exposure to calcineurin inhibitors.

Materials and Methods: Our study included 17 recipients of ABO-incompatible kidney transplant procedures performed at our institution. At baseline and at 3 and 12 months after conversion, graft biopsies were performed to check for acute rejection and C4d deposition.

Results: Treatment with everolimus was stopped due to adverse events in 8 patients (47.1%). Conversion to everolimus with calcineurin inhibitor minimization did not induce acute rejection or C4d deposition at 3 and 12 months after conversion in ABO-incompatible kidney transplant recipients in whom everolimus was maintained or stopped within 1 year after conversion.

Conclusions: Everolimus elicited no acute rejection and no C4d deposition, whether everolimus was maintained or stopped within 1 year after conversion, in ABO-incompatible kidney transplant recipients.

Key words : End-stage renal disease, Mammalian target of rapamycin inhibitor, Renal transplantation

Kidney transplant is the preferred treatment for end-stage renal disease (ESRD) because of its association with greater longevity and better quality of life compared with dialysis.¹ The shortage of deceased donors is a critical problem in Japan. To solve this problem, ABO-incompatible kidney transplant has been performed since the late 1980s in Japan. Excellent outcomes have been achieved, and the rates of graft survival in these patients are similar to those in recipients of ABO-compatible grafts.^2,3 Currently, ABO-incompatible kidney transplant is an acceptable renal replacement therapy option for patients with ESRD.^4-6 In ABO-incompatible kidney transplant, a desensitization protocol, consisting of a calcineurin inhibitor (CNI), rituximab, mycophenolate mofetil (MMF), and plasmapheresis, has been widely used in Japan. However, the high intensity of this protocol can lead to an increased rate of virus infection, including cytomegalovirus (CMV) and BK virus infections, in these transplant recipients.^7,8 Furthermore, MMF treatment is often discontinued due to adverse effects such as intractable diarrhea and CMV infection.

Everolimus is a mammalian target of rapamycin inhibitor with potent immunosuppressive activity. Everolimus is a safe alternative to standard immunosuppression therapy with CNIs and MMF.9 There have been few reports on the application of everolimus in ABO-incompatible kidney transplant. Our previous study demonstrated that conversion from MMF plus a standard-dose CNI regimen to everolimus plus low exposure to CNIs in stable ABO-incompatible kidney transplant patients was safe and effective during a 3-month observation period.¹⁰ Here, we present our 1-year follow-up data.

Materials and Methods

Patients
An open-label design was used to examine the safety of conversion of stable ABO-incompatible kidney transplant recipients from MMF with a standard-exposure CNI to everolimus with a low-exposure CNI regimen. In this prospective study, 17 recipients of ABO-incompatible kidney transplant procedures at our institution were included. The inclusion criteria for conversion were as follows: (1) at least 1 year posttransplant, (2) normal or slightly impaired renal function defined as a serum creatinine value < 2.0 mg/dL, (3) had received rituximab as desensitization protocol, (4) no acute rejection episodes for more than 6 months, (5) stable renal function for at least the prior 6 months, and (6) normal or slightly increased albuminuria defined as urinary albumin excretion (the ratio of spot urine albumin to creatinine) of < 100 mg/g creatinine. All patients were required to be receiving CNIs with MMF and steroids. The CNIs (cyclosporine or tacrolimus) were administered to achieve a blood trough level of 100 to 120 ng/mL for cyclosporine or 4 to 6 ng/mL for tacrolimus. For all patients, MMF and methlprednisolone were administered at 1 g/day and 4 mg/day, respectively.

Methods
At study entry, all patients received a graft biopsy to exclude acute cellular rejection and antibody-mediated rejection. All histologic findings were categorized according to the Banff 09 classification. We then analyzed the C4d staining results for all biopsy specimens. C4d staining was performed by immunofluorescence on frozen sections.

We have previously reported the protocol for this study. Briefly, patients without acute cellular rejection or antibody-mediated rejection diagnosed by graft biopsy were switched from MMF at 1 g/day to everolimus 1.5 mg/day in patients who received cyclosporine as CNI11 or everolimus 3.0 mg/day in patients who received tacrolimus as CNI,¹² with dose adjustments from 1 week onward to target an everolimus trough level of 3 to 8 ng/mL. The CNI dose was reduced to 40% to 60% below baseline values, with dose adjustments from 1 week onward to a targeted trough level of 25 to 50 ng/mL for cyclosporine or 2 to 4 ng/mL for tacrolimus. Baseline doses of methylprednisolone were continued unaltered in all patients. All adverse events were collected. The probability of freedom from discontinuation of everolimus administration was examined. Everolimus trough levels were assessed at 3, 6, and 12 months after conversion in patients in whom everolimus was maintained.

At 3 and 12 months after conversion, graft biopsies were performed to check for acute rejection and/or antibody-mediated rejection, whether everolimus was continued or discontinued. The morphologic features and C4d peritubular capillary staining were evaluated, and occurrences of acute rejection and/or antibody-mediated rejection episodes were analyzed. At baseline and at 3, 6, and 12 months after conversion, peripheral blood lymphocyte subsets (CD19 positive and CD20 positive) were monitored.

At baseline, clinical parameters (including age, sex, cause of ESRD, duration of dialysis, time to relative transplant, donor age, donor relation, ABO incompatibility, number of HLA mismatch antigens, and preoperative anti-blood-type antibody titers) were collected. At baseline and at 3, 6, and 12 months after conversion, fasting blood samples were obtained in the early morning for biochemical studies, including serum creatinine, fasting plasma glucose, total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and trough levels of CNIs. Estimated glomerular filtration rate (eGFR) was calculated using the modified Modification of Diet in Renal Disease equation using the new Japanese coefficient.13 Urinary albumin excretion was measured at baseline and at 3, 6, and 12 months after conversion. The effects of everolimus plus CNI minimization on renal function, urinary albumin excretion, and lipid profiles were examined.

All patients provided written informed consent before study enrollment, which was approved by the Human Ethics Committee of Osaka City University Hospital (Osaka, Japan). All procedures were in accordance with the Helsinki Declaration of 1975.

Statistical analyses
All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). More precisely, it is a modified version of R commander, which was designed to add statistical functions frequently used in biostatistics. The results are presented as mean values and standard deviations or as proportions for categorical variables. Changes were evaluated using paired t test or Wilcoxon test. Statistical significance was defined as P < .05.

Baseline characteristics
The mean age at transplant was 52.1 ± 14.7 years, and the mean time from transplant to conversion was 39.8 ± 22.9 months, with a range between 14 and 75 months posttransplant. Thirteen patients had received cyclosporine treatment, and 4 patients had received tacrolimus treatment. All patients received MMF 1 g/day and methylprednisolone 4 mg/day. The mean baseline values of serum creatinine and eGFR were 1.22 ± 0.21 mg/dL and 46.6 ± 5.9 mL/min/1.73 m², respectively. Other clinical characteristics of participants at baseline are shown in Table 1.

Adverse effects and discontinuation of everolimus
Treatment with everolimus was stopped due to adverse events in 8 patients (47.1%). Median time from conversion to discontinuation was 124.5 days, with a range from 109 to 271 days (Figure 1). Discontinuation of everolimus occurred 3 months after conversion. No death and graft loss occurred during the study. Adverse events were categorized as drug related when they occurred after conversion, with the most frequent events being hypercholesterolemia (70.5%), peripheral edema (47.1%), albuminuria (35.3%), aphthous stomatitis (17.6%), general fatigue (29.4%), anemia (5.9%), menoxenia (11.8%), and interstitial pneumonitis (5.9%). Five patients with general fatigue, 1 with interstitial pneumonia, 1 with peripheral edema, and 1 with menoxenia required discontinuation of everolimus and reconversion to MMF.

Histology and C4d staining at baseline and at 3 and 12 months after conversion
Histologic findings revealed no acute cellular rejection and no antibody-mediated rejection at baseline in any of the study patients. When the C4d staining patterns on the frozen sections were investigated, no patients showed diffuse C4d-positive staining. Focal C4d-positive staining and C4d-negative staining in the allograft biopsies were seen in 3 and 14 patients, respectively. Because both focal and negative C4d staining results were diagnosed as “C4d-negative,” there was no C4d deposition in any of the allograft biopsies for all recipients at baseline. One patient declined having a graft biopsy at 3 and 12 months after conversion. At 3 and 12 months after everolimus conversion, there were no reported acute cellular rejections or antibody-mediated rejections in the allograft biopsies. C4d staining patterns showed no diffuse C4d-positive staining (Table 2). Conversion to everolimus with CNI minimization did not induce acute rejection or C4d deposition along peritubular capillaries in the graft biopsies at 3 and 12 months after conversion in ABO-incompatible kidney transplant recipients in whom everolimus was maintained or stopped within 1 year after conversion.

Change in peripheral blood CD19-positive and CD20-positive cells
The respective mean percentage of peripheral blood CD19-positive and CD20-positive cells in the recipients enrolled in this study was 2.3 ± 4.9% and 3.4 ± 5.2% at baseline, 2.5 ± 4.5% and 3.8 ± 5.4% at month 3, 2.3 ± 4.4% and 3.0 ± 5.2% at month 6, and2.3 ± 4.6% and 3.4 ± 5.7% at month 12. In addition, there were no significant changes in the percentage of peripheral blood CD19-positive and CD20-positive cells during the study period.

Renal function
The mean eGFR value was 46.0 ± 5.7 mL/min/1.73 m² at baseline, 51.8 ± 7.5 mL/min/1.73 m² at month 3, 49.1 ± 7.0 mL/min/1.73 m2 at month 6, and 50.7 ± 7.8 mL/min/1.73 m² at month 12. Conversion from MMF to everolimus resulted in a significant but temporary elevation of eGFR at month 3. Three months after conversion, the mean eGFR did not significantly increase or decrease (Figure 1).

Albuminuria
The average urinary albumin excretion level increased significantly 6 months after conversion. Baseline albuminuria was 16.6 ± 13.5 mg/g creatinine, increasing to 32.4 ± 33.6 mg/g creatinine at month 3 and to 30.4 ± 18.7 mg/g creatinine at month 6. However, urinary albumin excretion did not show a significant change between baseline and month 12 (Figure 1).

Change in doses and trough concentrations of everolimus and calcineurin inhibitors
There were no significant differences in everolimus trough concentrations and CNI trough concentrations between patients who continued everolimus and those who discontinued everolimus (data not shown).

In this study, we found that everolimus with CNI minimization elicited no acute rejection, suppressed B-cell function and activation, and induced no deterioration of renal function in ABO-incompatible kidney transplant recipients, whether everolimus was maintained or stopped within 1 year after conversion. This study suggests that there may be no disadvantage for ABO-incompatible kidney transplant recipients to try converting from MMF to everolimus to avoid adverse events associated with MMF, such as gastrointestinal complications, intractable CMV infection, and BK virus activation. These results demonstrated that conversion from MMF to everolimus may be a safe alternative for ABO-incompatible kidney transplant recipients.

There have been few reports on application of everolimus regimens for ABO-incompatible kidney transplant recipients. In their study, Koch and associates reported on ABO-incompatible kidney transplant recipients with an immunosuppressive regimen based on de novo everolimus and low-dose CNI, finding that the number of viral infections was clearly below the expected range.¹⁴Recently, Belliere and associates demonstrated that ABO-incompatible kidney transplant recipients with an active BK
virus infection may benefit from conversion to everolimus.¹⁵ In our present study, we revealed that everolimus with CNI minimization elicited no acute rejection and no C4d deposition whether everolimus was maintained or stopped within 1 year after conversion in ABO-incompatible kidney transplant patients. Everolimus may be a possible alternative for viral infection in ABO-incompatible kidney transplant recipients.

In a recent report, although mycophenolic acid and everolimus efficiently suppressed cell proliferation during the early phase of B-cell immune reaction, everolimus was shown to act in a later phase versus mycophenolic acid.16 Furthermore, a mammalian target of rapamycin inhibitor was the most effective drug in inhibiting B-cell activation compared with cyclosporine, tacrolimus, and mycophenolic acid.¹⁷ For ABO-incompatible kidney transplant patients, the immunosuppressive protocol requires inclusion of an effective B-cell-targeted therapy to prevent antibody-mediated rejection by anti-A/B antibody titer. These finding may support our results that everolimus with CNI minimization suppressed peripheral blood CD19-positive and CD20-positive cells during the study period.

In another study, although the overall incidence of adverse events was similar in everolimus and mycophenolic acid treatment groups, the overall incidence of adverse events leading to drug discontinuation was significantly higher in the everolimus treatment group.11 In addition, adverse events of mammalian target of rapamycin inhibitors accounted for a 20% to 40% dropout rate in a clinical phase 3 trial.18 In our present study, treatment with everolimus was stopped due to adverse events in 8 patients (47.1%) 3 months after conversion, which is similar to that shown in the literature regarding ABO-incompatible kidney transplant recipients experiencing adverse events and everolimus discontinuation.

At 3 months after conversion, we found that everolimus with CNI minimization induced a significant but short-term increase in eGFR.10 Those recipients who required everolimus discontinuation at 3 months after conversion may demonstrate a lack of benefit regarding renal function. In our small sample size, even recipients who were maintained on everolimus showed no improvements in renal function at month 12 after conversion.

Urinary albumin excretion was significantly elevated at month 3 and month 6 after conversion to everolimus in this study. However, this phenomenon was transitory, and urinary albumin excretion at month 12 was overall similar to that at baseline. Our results showed that elevation of urinary albumin excretion due to conversion to everolimus was decreased by angiotensin-receptor blocker administration or everolimus discontinuation.

Our present study has several limitations, including its small sample size and short follow-up duration. Despite these limitations, most of the recipients enrolled in this study received graft biopsies before and after termination of the study. Our results indicated the safety of conversion from MMF with standard exposure to CNIs to everolimus with low exposure to CNIs in ABO-incompatible kidney transplant recipients whether everolimus was maintained or stopped within 1 year after conversion.

In conclusion, our study showed that everolimus with CNI minimization elicited no acute rejection, suppressed B-cell function and activation, and induced no deterioration in renal function in stable ABO-incompatible kidney transplant recipients. In cases of gastrointestinal complications, intractable CMV infection, and BK virus activation, conversion to everolimus may be a safe alternative in ABO-incompatible kidney transplant recipients. To assess the safety of everolimus for ABO-incompatible kidney transplant in the long term, further prospective well-controlled, long-term follow-up trials with a larger number of patients are needed.

Volume: 17 Issue: 2 April 2019

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