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Early Immunosuppressive Exposure of Enteric-Coated-Mycophenolate Sodium Plus Tacrolimus Associated with Acute Rejection in Expanded Criteria Donor Kidney Transplantation

Background: Immunosuppressive agents are still inefficient in preventing biopsy-proven acute rejection (BPAR) after expanded criteria donor (ECD) kidney transplantation. The aim of this study was to investigate the relationships between early immunosuppressive exposure and the development of BPAR. Methods: We performed a retrospective study of 58 recipients of ECD kidney transplantation treated with enteric-coated-mycophenolate sodium, tacrolimus (Tac), and prednisone. The levels of mycophenolic acid-area under the curve (MPA-AUC)0-12h and Tac C0 were measured at the 1st week and the 1st month posttransplant, respectively. The correlation was assessed by multivariate logistic regression. Results: The occurrence rates of BPAR and antibody-mediated rejection were 24.1% and 10.3%, respectively. A low level of MPA-AUC0-12h at the 1st week posttransplant was found in BPAR recipients (38.42 ± 8.37 vs. 50.64 ± 13.22, P < 0.01). In addition, the incidence of BPAR was significantly high (P < 0.05) when the MPA-AUC0-12h level was <30 mg·h-1·L-1 at the 1st week (15.0% vs. 44.4%) or the Tac C0 was <4 ng/ml at the 1st month posttransplant (33.3% vs. 21.6%). Multivariable logistic regression analysis showed that the MPA-AUC0-12h at the 1st week (OR: 0.842, 95% CI: 0.784-0.903) and the Tac C0 at the 1st month (OR: 0.904, 95% CI: 0.822–0.986) had significant inverse correlation with BPAR (P < 0.05). Conclusions: Low-level exposure of MPA and Tac C0 in the early weeks posttransplant reflects an increased acute rejection risk, which suggested that MPA-AUC0-12h <30 mg·h-1·L-1 and Tac C0 <4 ng/ml should be avoided in the first few weeks after transplantation.

cardiovascular risk, and malignancies. [5,6] For this reason, the ideal immunosuppressive regimen for this population has not yet been defined. Therefore, the aim of this study was to assess the relationship between early immunosuppressive exposure and BPAR in DCD-ECD kidney transplant recipients and to provide a reasonable basis for clinical application.

Ethical approval
This is a single-center, retrospective, observational cohort study approved by the local institutional review board of the First Affiliated Hospital of Xi'an Jiaotong University, which was in compliance with the provisions of the current Declaration of Helsinki principles and Good Clinical Practice guidelines. Written informed consent was obtained from all participants.

Design
All patients who received an ECD kidney-only first transplant between July 2012 and June 2016 were included in this study. Follow-up was until May 2017. End points studied were as follows: (1)

Clinical definitions
ECDs were identified according to the United Network for Organ Sharing definition (age ≥60 years or 50-59 years with at least two of the following: hypertension, death from cerebrovascular accident, and terminal creatinine ≥132 µmol/L). [4] The diagnostic criterion for DGF is dialysis needed in the 1 st week after kidney transplantation. Allograft biopsy was performed for suspected acute rejection (AR) cases. AR was identified on biopsy and classified according to the Banff 2013 classification. [7] Graft failure was established when long-term dialysis treatment was reestablished or upon death.

Immunosuppressive regimen
A triple immunosuppressive regimen of enteric-coatedmycophenolate sodium (EC-MPS; myfortic ® , Novartis, Basel, Switzerland), tacrolimus (Tac), and prednisone (pred) was the initial maintenance immunosuppressive regimen used in all patients. The initial EC-MPS dose was 1440 mg/d administered within 24 h posttransplantation; Tac was administered at 0.06 mg·kg −1 ·d −1 beginning on the 3 rd day after transplantation. The target Tac C 0 level was 4-10 ng/ml from the beginning of transplantation. Oral pred was administered at 10 mg/d after transplantation. The dosage of immunosuppressive agents was adjusted according to clinical experience, biochemical results, and effective exposure of the drug. All recipients were induced with rabbit antithymocyte globulin (ATG; Thymoglobulin ® ; Genzyme, Waterford, Ireland; 1.25 mg·kg −1 ·d −1 between days 0 and 4 after transplantation).
Tacrolimus C 0 monitoring and pharmacokinetic assessment of enteric-coated-mycophenolate sodium Blood samples for Tac C 0 were drawn before morning medication. All samples were anticoagulated with ethylenediaminetetraacetic acid. Whole-blood Tac concentration levels were measured using a fluorescence polarization immunoassay on an AxSYM analyzer (Abbott Diagnostic, Chicago, IL, USA). The mycophenolic acid (MPA) concentration levels were measured by an enzyme-multiplied immunoassay technique (Mycophenolic Acid Assay; Siemens Healthcare Diagnostic, Camberley, UK) at the 1 st week and 1 st month posttransplantation. Blood samples were collected before the morning medication and at 0

Management of biopsy-proven acute rejection
BPAR cases were treated with 500 mg methylprednisolone administered intravenously for 3 consecutive days combined with optimized Tac and EC-MPS therapy. ATG was administered for 5-10 days for steroid-resistant and early high-grade ARs. [8]

Statistical analysis
The Chi-square test was used to analyze categorical data. Student's t-tests were used as appropriate for continuous data. Multivariate logistic regression analysis was used to determine the correlation between MPA-AUC 0-12h and Tac C 0 level in the early weeks and BPAR during first 12 months posttransplant; A value of P < 0.05 was considered statistically significant. SPSS version 19.0 (SPSS Inc., Chicago, Illinois, USA) was used for statistical analysis.

Graft outcomes
Fifty-eight patients who received kidney grafts from DCD-ECD donors during the study time period were enrolled in this study. The average follow-up time was 10.7 month (range: 0.8-12.0 months). Demographic and clinical characteristics of the donors and recipients are shown in Tables 1 and 2. The incidence of BPAR and antibody-mediated rejection was 24.1% and 10.3%, respectively. DGF was observed in 13 patients (22.4%). The allograft function of eGFR was 58.4 ± 13.9 ml·min −1 ·1.73 m −2 at 12 months posttransplant. The graft survival and patient survival were 86.2% and 89.7%, respectively, in the 12 months following kidney transplantation [ Table 3]. The graft loss (n = 8) included rejection unrevised (n = 3), DGF was unretired (n = 3), renal artery stenosis (n = 1), and chronic allograft nephropathy (n = 1). The cause of death (n = 6) included cardiovascular disease (n = 3), multiple organ dysfunction syndrome (n = 2), and lung infection (n = 1).

Mycophenolic acid exposure and the risk of biopsy-proven acute rejection
It had been shown that the BPAR was associated with the dose of MPA and its exposure. To definitively prove this, we percentage of MPA-AUC 0-12h <30 mg·h -1 •L -1 at the 1 st month was significantly lower than at the 1 st week (31.0% vs. 8.8%, P < 0.01), whereas the MPA-AUC 0-12h of 30-60 mg·h -1 •L -1 at the 1 st month was significantly higher than that at the 1 st week (P < 0.01) [ Figure 1d].

Tacrolimus C 0 and the risk of biopsy-proven acute rejection
We analyzed the relationship between the Tac dosage and BPAR. Our data showed that the Tac dosage was similar in the BPAR and non-BPAR groups during the study period [ Figure 2a], but the Tac C 0 level in the BPAR group was significantly lower than that in the non-BPAR group at the 1 st month posttransplantation (P < 0.05) [ Figure 2b]. We used the cut-points Tac C 0 <4.0 versus ≥4.0 ng/ml and Tac C 0 <7.0 versus ≥7.0 ng/ml to analyze their association with the BPAR rate and found that the incidence of BPAR was significantly higher when Tac C 0 was <4 ng/ml compared with Tac C 0 ≥4.0 ng/ml at the 1 st month posttransplant (7.2 ± 1.2 vs. 8.3 ± 2.7, P < 0.05) [ Figure 2c]. This phenomenon recurred when Tac C 0 <7.0 versus ≥7.0 ng/ml at the 1 st week and 1 st month after transplantation, although it was not statistically significant [ Figure 2d].

Association analysis between mycophenolic acid-area under the curve 0-12h /tacrolimus C 0 and biopsy-proven acute rejection
To better identify the relationship between immunosuppressive exposure and BPAR, we built a multivariate logistic regression model including all the variables of MPA-AUC 0-12h and Tac C 0 at the 1 st week and 1 st month posttransplant. As shown in Table 4, the MPA-AUC 0-12h at the 1 st week posttransplant (odds ratio [OR], 0.842, 95% confidence interval [CI], 0.784-0.903, P < 0.05) and the Tac C 0 at the 1 st month posttransplant (OR, 0.904, 95% CI, 0.822-0.986) were identified as an independent risk factor of BPAR (P < 0.05). These results indicated that MPA-AUC 0-12h <30 mg·h -1 •L -1 and Tac C 0 <4.0 ng/ml in the early weeks had a significant correlation with AR. dIscussIon ECD transplantations are complicated by increased rates of DGF and AR, especially in the earlier posttransplantation period, and an adequate level of immunosuppressant is desired under these circumstances. Recipients of ECD kidneys were often excluded from transplant trials, and therefore the optimal maintenance regimen for them is unknown. This single-center study retrospectively followed 58 ECD kidney transplant recipients and investigated the association between Tac C 0 and MPA-AUC 0-12h and AR risk during the first 12 months posttransplant.
For all the study recipients, there was a significant correlation between the MPA-AUC 0-12h and the risk of AR after renal transplantation. These results are consistent with the studies that suggest an inverse association between MPA exposure and a risk of AR. [9,10] Adequate MPA exposure in the early posttransplant phases is required because patients are at the highest risk of AR during this period. A therapeutic window divided the patients into two groups: BPAR and non-BPAR and analyzed the relationship between EC-MPS dose and its exposure with BPAR. The dose of EC-MPS was similar in the two groups during the study period [ Figure 1a], but the MPA-AUC 0-12h in the BPAR group was significantly lower than that in the non-BPAR group at the 1 st week posttransplantation (38.42 ± 8.37 vs. 50.64 ± 13.22, P < 0.01) [ Figure 1b]. Further analysis showed that the incidence of BPAR was significantly increased when the MPA-AUC 0-12h was <30 mg·h -1 •L -1 at the 1 st week posttransplant (15.0% vs. 44.4%, P < 0.05) [ Figure 1c]. After comparing the MPA-AUC 0-12h range in the 1 st week and 1 st month posttransplant, we found that the  of MPA-AUC has been recommended (30-60 mg·h -1 •L -1 ) to achieve optimal efficacy. [10,11] Similarly, our data showed that the incidence of BPAR was significantly higher when MPA-AUC 0-12h was <30 mg·h -1 •L -1 compared with ≥30 mg·h -1 •L -1 at the 1 st week posttransplant in DCD-ECD kidney transplant patients (P < 0.05). Moreover, to better identify the association between MPA exposure and BPAR, we built a multivariate logistic regression model including MPA-AUC 0-12h (ref <30 mg·h -1 •L -1 ) at the 1 st week and 1 st month posttransplant. These results suggest that MPA-AUC 0-12h <30 mg h/L at the 1 st week has a significant correlation with AR during the first 12 months after transplantation (P < 0.05). However, the increase in the AR rate was similar in recipients with an MPA-AUC 0-12h <30 mg h/L or ≥30 mg h/L (20.0% vs. 23.1%) at the 1 st month. After analyzing the range of MPA-AUC 0-12h , we found that the compliance rate was significantly higher at the 1 st month than at the 1 st week posttransplant (P < 0.01). This implies that the presence of significant difference during the first 12 months is largely due to the differences that were already achieved before the 1 st month. [12] As a result of the extensive application of MPA and CNIs, many studies have demonstrated that CNI exposure can cause acute and chronic nephrotoxicity; thus, low exposure to CNIs has been advocated in the recent years to further improve transplant outcomes, particularly in ECD kidneys. [13][14][15][16][17] However, many studies have shown  58.4 ± 13.9 Data was presented as mean ± SD or n (%). AR: Acute rejection; AMR: Antibody-mediated rejection; BPAR: Biopsy-proven acute rejection; DGF: Delayed graft function; eGFR: Estimated glomerular filtration rate.  that lower Tac C 0 levels during the 1 st week, [18] during the 1 st month, [19] after 3 months, [20] and Tac C 0 of <4 ng/ml [13] or <7 ng/ml [21] posttransplant were significantly correlated with the subsequent higher BPAR rates. Therefore, we decided to use the cut-point <4.0 versus ≥4.0 ng/ml and <7.0 versus ≥7.0 ng/ml to analyze the association between Tac C 0 and BPAR risk during the first 12 months after transplantation.
In our results, the percentage of <4 ng/ml was 22.4 and <7 ng/ml was 34.5 at the 1 st week posttransplantation. In addition, the percentage of <4 ng/ml was 15.5 and <7 ng/ml was 20.7 at the 1 st month posttransplantation. The incidence of BPAR was significantly higher when Tac C 0 was <4 ng/ml compared with Tac C 0 ≥4.0 ng/ml at the 1 st month posttransplantation (P < 0.05). This phenomenon recurred when Tac C 0 <7.0 versus ≥7.0 ng/ml at the 1 st week and 1 st month after transplantation, although it was not statistically significant. The results provide a more accurate description of the correlation between lower Tac C 0 levels and subsequent higher BPAR risk, and notably 4.0 ng/ml represented the minimum of the target Tac C 0 level range specified in our protocol at the 1 st month after transplantation. Research indicates that lower MPA-AUC values were associated with a significantly higher BPAR risk during the first 12 months after transplantation. Reanalysis of the Opticept [22] and FDCC [12] trials supports a lower MPA-AUC at day 3 after transplantation which was associated with a significantly higher AR rate during the first 12 months. However, none of these analyses include the prognostic influence of CNI level, either tested or controlled. More recently, Daher Abdi et al. [10] reported that lower MPA-AUC was more significantly associated with subsequently higher AR risk than a lower Tac level. In fact, our findings further confirm their conclusion.
Currently, the most common immunosuppressive treatment after de novo renal transplantation is a triple regimen including Tac, MPA, and corticosteroids. In addition, most centers use antibody induction systematically in selected patients at high immunologic risk; for example, ECD kidney, PRA positive. In many recent experimental regimens, and increasingly in clinical practice also, the recommended target concentration for Tac is <10 ng/ml, which is lower than that in previous years, as reflected by the newly revised prescribing information in the United States (recommended target concentration 4-11 ng/ml in combination with MPA and an interleukin-2 receptor antagonist). [23] For ECD kidney transplantation, we recommended Tac target concentration 4-10 ng/ml in combination with MPA and rabbit ATG. In addition, let MPA reach adequate exposure early.
In conclusion, low-level exposure to MPA and Tac C 0 in the early weeks posttransplantation reflects an increased AR risk, which suggests that MPA-AUC 0-12h <30 mg h/L and Tac C 0 <4 ng/ml should be avoided in the first few weeks after transplantation.

Conflicts of interest
There are no conflicts of interest.