Key words : Interstitial fibrosis/tubular atrophy, Mycophenolate mofetil, Renal transplantation

Introduction

Kidney allografts that were previously lost because of acute rejection or early patient death are now lost to other harmful mechanisms. Late allograft failure that is censored for death can be subdivided mainly by cause as follows: (1) glomerular disease, including recurrent/new glomerulonephritis disease, and (2) interstitial fibrosis and tubular atrophy without any specific etiology (IF/TA).¹ Specific causes of IF/TA have been identified as acute rejection, vascular remodeling, calcineurin inhibitor (CNI)-induced nephrotoxicity, and cytomegalovirus (CMV) infection.^2-4 However, IF/TA has been rarely attributed solely to CNI toxicity alone.1 In addition, recent modest tacrolimus dosing regimens have been associated with reduced nephrotoxic effects compared with previous high-dose cyclosporine regimens.⁵

Everolimus is an immunosuppressive drug that has been classified as an inhibitor of the mechanistic target of rapamycin (mTOR); mTOR inhibitors block growth factor-mediated cell proliferation, suppress T-cell activation, and exert potent immunosup-pression in transplant recipients.⁶ Compared with mycophenolate mofetil (MMF), everolimus exhibits antineoplastic, antiviral, antiatherosclerosis, and antiproliferative properties. The beneficial effects of mTOR inhibitors on renal fibrosis have also been suggested.⁷

In 2016, our institute introduced an everolimus-based protocol for all patients with low immuno-logical risk who received de novo living donor kidney transplant (KT). Because CNI doses have decreased over the past 15 years, it is unknown whether additional reductions in CNI exposure are still needed for patients who receive everolimus compared with MMF; thus, we adopted a protocol in which everolimus is combined with standard-dose tacrolimus before administration of combination therapy with low-dose tacrolimus. The aim of the present study was to compare the outcomes of everolimus plus standard tacrolimus immunosup-pression versus the outcomes MMF plus standard tacrolimus immunosuppression in recipients of de novo living donor KT.

Materials and Methods

Ethics approval and informed consent
All procedures performed involving human participants were conducted in accordance with the ethical standards of our institutional and national research committees (IRB-No 24-54/UMIN000008475) and were performed in accordance with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

Because data were retrospectively obtained from medical records, the institutional review board waived the requirement for informed consent from patients in accordance with the ethical guidelines for medical and health research involving human subjects in Japan.

Patients
Our study included patients who received KT between January 2013 and December 2018. Patient data were extracted from medical records at our facility. In September 2016, we introduced an everolimus-based protocol for all low immunological risk patients who underwent de novo KT. Therefore, almost all patients in the MMF group underwent KT from January 2013 to August 2016. However, almost all patients in the everolimus group received KT from September 2016 to December 2018. During the study period for each group, the results at 1 year were compared between the 2 groups. In addition, all patients were considered to have a low immuno-logical risk based on the following exclusion criteria: (1) deceased donor KT; (2) pediatric patients less than 18 years old; (3) patients with antiphospholipid syndrome; (4) patients with preformed donor-specific anti-HLA antibody; (5) patients with ABO-incom-patible transplant; and (6) retransplant recipients.

Immunosuppression protocol
All patients who underwent KT received a once-daily prolonged-release formulation of tacrolimus and methylprednisolone orally; they also received basiliximab (20 mg/day) at the time of surgery and on day 4 posttransplant. In the everolimus group, everolimus was started at 2.0 mg/day (from day 7 pretransplant), with dose adjusted to achieve target trough concentrations of 3 to 8 ng/mL throughout.⁸ In the MMF group, MMF was started at 2.0 g/day (from day 7 pretransplant). The dose was reduced to 1.5 g/day after 2 months and 1 g/day after 3 months. The once-daily prolonged-release formulation of tacrolimus was started at 0.10 mg/kg/day (from day 7 pretransplant). The whole blood trough concen-tration was maintained at 5 to 8 ng/mL for 1 or 2 months after transplant and then adjusted to maintain a trough concentration of 4 to 8 ng/mL. Methylprednisolone (250 mg) was administered intravenously on the day of transplant (Figure 1a).

Study variables and definitions
The following demographic and clinical data were collected retrospectively from medical records: renal function, urine protein, patient death, allograft loss, biopsy-proven acute rejection, BK virus-associated nephropathy, surgery complications, infections (including CMV antigenemia), delayed graft function, newly developed posttransplant diabetes mellitus, hyperlipidemia, leukopenia, and anemia. To evaluate renal function, the estimated glomerular filtration rate (eGFR) was calculated using an appropriate equation for Japanese patients with chronic kidney disease.⁹ Urinary protein was assessed using the spot urine protein-to-creatinine ratio at 6 and 12 months.

Episode biopsies or 3-month and 1-year protocol biopsies were performed on allografts, and the diagnosis was made in accordance with the Banff 2019 working classification.¹⁰ Patients with acute rejection were classified as follows: borderline changes, acute T-cell-mediated rejection (Banff grade 1A or higher), and/or acute antibody-mediated rejection. BK virus-associated nephropathy and allograft rejection were restricted to biopsy-proven diagnoses. All biopsy specimens were evaluated by 2 experienced nephrologists (A.T. and K.U.) who reached a consensus using a dual light microscope.

Postoperative complications were graded in accordance with the Clavien-Dindo classification,11 with grade ≥3 indicating the presence of pos-toperative complications. Cytomegalovirus infection was defined as detection of CMV replication based on the presence of CMV pp65 antigenemia (≥10 CMV pp65-positive cells per 200 000 peripheral blood leukocytes). Delayed graft function was defined as a need for dialysis therapy within 1 week of transplant despite the diagnosis determined via biopsy. New-onset posttransplant diabetes mellitus was diag-nosed in accordance with the American Diabetes Association definition.¹² Leukopenia, anemia, and hyperlipidemia were assessed by white blood cell count and hemoglobin, serum triglyceride, and low-density lipoprotein levels at 6 and 12 months.

Statistical analyses
Results are presented as means ± standard deviations or as counts and percentages for categorical variables. Mean bivariate differences between the 2 groups were assessed using t tests. Grades of IF/TA were compared using the 2-sided Mann-Whitney U test. Categorical variables were compared using the chi-square test or the Fisher exact test. To overcome bias from different distributions of covariables among patients in the 2 study groups, inverse probability of treatment weighting (IPW) was performed using logistic regression analysis to generate propensity scores for both groups of patients. The following variables that affected the primary outcomes were included in the IPW analysis: recipient age/sex, dialysis duration, CMV mismatch (seronegative recipient and seropositive donor), cause of end-stage renal disease, donor age/sex, and number of HLA mismatches. Allograft survival and rejection-free survival rates were calculated using the Kaplan-Meier method, and differences between curves were evaluated using the log-rank test. Multivariate ordered logistic regression analysis was performed to identify the risk factors for grades of IF/TA. All statistical analyses were performed using JMP Pro 16 (SAS Institute Inc). P < .05 was considered statistically significant.

Results

Patient characteristics
From January 2013 to December 2018, 469 consecutive patients underwent KT at Kyushu University Hospital (Fukuoka, Japan). Among them, 225 patients met the criteria for inclusion into this study (Figure 1b). All living donors were relatives of the recipient (within fourth-degree relatives, spouses, or second-degree relatives-in-law). The donors included parents for 100 patients (44.4%), spouses for 68 patients (30.2%), siblings for 40 patients (17.8%), children for 12 patients (5.3%), an uncle for 1 patient (0.4%), a first cousin for 1 patient (0.4%), spouse’s parents for 2 patients (0.9%), and a spouse’s sibling for 1 patient (0.4%). The baseline characteristics of each group (before and after IPW) are shown in Table 1. No significant differences were observed between the 2 groups. The mean age of each group was 48 ± 13 years in the everolimus group and 46 ± 14 years in the MMF group (P = .304). The female-to-male ratios were 28/56 in the everolimus group and 46/95 in the MMF group (P = .913). Before IPW, only the dialysis duration was significantly longer in the MMF group compared with the everolimus group (P = .001). After IPW, there were 85 patients in the everolimus group and 141 patients in the MMF group, with characteristics again statistically similar. After IPW, 12 patients (14%) in the everolimus group deviated from the protocol at year 1 compared with 22 patients (16%) in the MMF group (P = .692).

Immunosuppression
In both groups, the mean tacrolimus trough concentration was within the target range throughout the study. However, tacrolimus trough concentration (C0) was significantly higher in the everolimus group than in the MMF group at all time points based on the t test (Figure 2a). In the everolimus group, the mean everolimus trough concentration was in the target range (3-8 ng/mL) throughout the study (Figure 2b).

Perioperative outcomes
Perioperative outcomes are summarized in Table 2. Warm ischemia time, the incidence of death, allograft loss, biopsy-proven acute rejection, BK virus-associated nephropathy, surgical complications (Clavien-Dindo grade ≥3), delayed graft function, and new-onset posttransplant diabetes mellitus were not significantly different between the 2 groups. There was a longer total ischemia time in the everolimus group compared with the MMF group (P < .001). There was a lower incidence of CMV antigenemia in the everolimus group compared with the MMF group (P < .001).

White blood cell count, hemoglobin, proteinuria, triglycerides, and low-density lipoprotein
The white blood cell count was significantly higher in the everolimus group compared with that shown before immunosuppressant administration was started, and this higher level remained at 6 months after KT. There were no significant differences in hemoglobin and the spot urine protein-to-creatinine ratio throughout the study. The mean triglycerides and low-density lipoprotein concentration at 6 and 12 months after KT were significantly higher in the everolimus group than in the MMF group (Table 3).

Graft function, graft survival, and rejection-free survival
Using t tests, we found that eGFR in the everolimus group was significantly lower than eGFR in the MMF group at all time points within 1 year after KT (Figure 2c). Death-censored allograft survival and rejection-free survival after KT were not significantly different between the groups (Figure 2d and Figure 2e).

Pathological outcomes
There was no significant difference in IF/TA severity in the 0-hour, 3-month, and 1-year protocol biopsies (Figure 2f). Multivariate ordered logistic analysis showed that older donor age and an acute rejection episode, but not everolimus or mean tacrolimus C0 throughout the first postoperative year, were significant risk factors for a higher IF/TA grade at the 1-year protocol biopsy (Table 4; P = .004 and P < .001, respectively).

Discussion

In this IPW study, we investigated the outcomes
of everolimus plus standard-dose tacrolimus in patients with de novo KT. The results suggested that everolimus with standard-dose tacrolimus resulted in similar outcomes for de novo KT recipients compared with the standard regimen with MMF.

In the ATHENA study¹³ (a 12-month, prospective, randomized controlled multicenter trial in de novo KT patients), the investigators compared everolimus versus MMF with similar tacrolimus exposure in both groups, or everolimus with concomitant tacrolimus, or cyclosporine. Based on results for eGFR, urinary protein, incidence of biopsy-proven acute rejection, BK virus-associated nephropathy, allograft loss, and death, the study reported that the everolimus-tacrolimus regimen was not inferior to the MMF-tacrolimus regimen. Furthermore, the study suggested that the everolimus-tacrolimus regimen was better for CMV suppression than the MMF-tacrolimus regimen.¹³ Similarly, in our study, we found no significant differences between the everolimus- and MMF-based regimens for urinary protein, incidence of biopsy-proven acute rejection, BK virus-associated nephropathy, and allograft loss and death, as well as a significantly lower incidence of CMV antigenemia in the everolimus group compared with the MMF group. However, there was a significant difference in the eGFR results between the 2 groups. The eGFR may have been different because there was a significant difference in tacrolimus C0 throughout the first year after transplant. An everolimus-based regimen has also been shown to suppress the BK virus-associated nephropathy as well as CMV antigenemia when combined with low-dose CNI.⁹ This suggests that everolimus alone does not have any advantage over MMF in the development of BK virus-associated nephropathy and suppresses it only when the dose of CNI is reduced.

The antiproliferative properties of mTOR inhibitors may interfere with the wound-healing process posttransplant.¹⁴ However, another study showed comparable risks of adverse effects on wound-healing events between everolimus- and MMF-based regimens. Our study showed that there were no differences in surgical complications between the 2 groups.⁸ In the everolimus group, total ischemia time was longer compared with that shown in the MMF group. This difference was due to the larger number of KTs that were performed at our institution after the introduction of the everolimus protocol; thus, donor and recipient surgery could not be performed simultaneously. However, the longer total ischemia time had no effect on outcomes for recipients. There was no significant difference between the 2 groups in terms of protocol deviations due to adverse effects that occurred during the first year after transplant. Therefore, the adverse effects appeared to be comparable between our 2 study groups.

Interstitial fibrosis/tubular atrophy is one of the most important factors for prognoses of allograft survival,¹⁵ but strategies to prevent interstitial renal fibrosis after KT remain unclear. In our study, ordered logistic regression models did not show a significant association between IF/TA severity at 1 year and the mean tacrolimus trough concentrations during the first year after transplant. Minimization of CNIs has been shown to be associated with a modest increase in renal function, but persistent damage is present in biopsies for the duration of CNI administration.¹⁶ However, the effect of serum tacrolimus concentration on short-term IF/TA in allografts is controversial.^1,17

Kidney fibrosis is primarily caused by the action of transforming growth factor β, which induces fibroblast proliferation and myofibroblast transition. It has been suggested that transforming growth factor β activates the Akt/mTOR complex 1 axis via the PI3K/Akt/TSC2-dependent pathway.¹⁸ Inhibition of mTOR complex 1 with rapamycin (an mTOR inhibitor) reduces the number of interstitial fibroblasts and myofibroblasts and decreases renal interstitial fibroblasts in obstructive nephropathy rodent models.⁷ However, our study could not show the superiority of the everolimus-based regimen in preventing IF/TA, but an allograft rejection episode was the most significant factor. This confirms that adequate immunosuppressive therapy is the most important strategy for IF/TA prevention that leads to long-term graft survival. Randomized clinical trials have confirmed that coadministration of everolimus plus reduced-exposure CNI therapy did not com-promise immunosuppressive efficacy.^8,19 In addition, everolimus therapy may also have non-immunosup-pressive benefits.^20,21 With the consideration that CMV suppression has been shown in regimens with everolimus and BK virus suppression has been shown in combination with low-dose CNI,⁹ induction regimens with everolimus may be the optimal option because they are expected to provide sufficient immunosuppression while preventing certain infections. Therefore, for immunologically low-risk KT, the combination of everolimus and reduced tacrolimus seems to be a better regimen to extract the benefits of everolimus.

Our study had several limitations. First was its retrospective observational study design. Although IPW was used, unmeasurable confounders may have affected our results. In addition, the 2 groups did not undergo KT during the same period. Patients in the everolimus group underwent KT more recently than patients in the MMF group. Although only patients with a low immunological risk were included in this study, the more recent KT procedure in the everolimus group may have contributed to the improved outcome compared with the MMF group. Therefore, there may have been a time period bias. Second, the observational period was short. Third, we did not assess de novo donor-specific anti-HLA antibody, which constitutes a potent risk factor for allograft survival.

Conclusions

Our study suggested that outcomes of an everolimus plus standard-dose tacrolimus regimen in recipients of de novo KT were comparable to those of a MMF and standard-dose tacrolimus regimen.

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