Donor CYP3A5 Expression Decreases Renal Transplantation Outcomes in White Renal Transplant Recipients

Background

Calcineurin inhibitors are among the most important groups of immunosuppressants, with tacrolimus (TAC) being the first-choice agent for the treatment of renal transplant recipients (RTRs) [1]. When TAC was placed on the market, it reduced biopsy-proven acute rejection (BPAR) occurrence at 12-month follow-up to 10–20%. Early BPAR is a crucial factor affecting overall patient and allogeneic graft survival [2,3].

TAC dosage adjustment remains a challenge owing to its narrow therapeutic index and high variability in pharmacokinetics. Underexposure favors allograft rejection, whereas overexposure results in severe adverse effects. Treatment requires therapeutic drug monitoring using whole-blood trough TAC concentration (C0) measures [1,4].

It is primarily metabolized by CYP3A cytochromes localized in the liver and gut mucosa. The function of CYP3A5 is well documented. The expression of CYP3A5 enzymes, which accelerate TAC metabolism and elimination, depends on the CYP3A5*1 coding allele, which is found in 10–15% of the White population. The common allelic polymorphism CYP3A5*3 (rs776746; 6986A>G) causes aberrant splicing and produces a protein lacking enzymatic function [4,5]. Thus, people who are CYP3A5 expressors are expected to have lower TAC blood concentrations than non-expressors. Numerous reports have confirmed that RTRs carrying at least 1 CYP3A5*1 allele (*1/*1 or *1/*3) have a lower TAC concentration-to-dose ratio than non-expressors (CYP3A5*3/*3). Genetic factors seem to play an important role in the dose and blood concentration variability of TAC [6–8].

Therefore, it was expected that the recipient’s CYP3A5 genotype may have an impact on renal function after transplantation, yet numerous studies and meta-analyses refuted that assumption [8–11]. On the other hand, CYP3A5 expression in renal tubular cells is well documented [12]. The significance of this phenomenon remains unclear, but an in vitro study showed that the concentrations of inactive metabolites of TAC in the kidney tissue of CYP3A5 expressors were significantly greater than in CYP3A5 non-expressors [13]. In addition, some reports have confirmed that RTRs receiving allogeneic kidney without CYP3A5 expression were at increased risk of TAC nephrotoxicity [14–17], emphasizing the role of its local clearance. Very few authors have investigated the influence of donor CYP3A5 expression on post-transplant allograft function, and the available studies present conflicting results [10,11,18–21].

Our cohort study aimed to assess whether the CYP3A5 genotype of the renal graft affects kidney survival and transplantation outcomes in the short- and long-term post-transplant periods.

Material and Methods

STUDY COHORT:

We conducted a retrospective cohort study in 95 deceased donor kidney recipients who were selected from among 595 patients who underwent transplantation between January 2010 and January 2017 at a single center. Patients were considered eligible for inclusion based on the following criteria: 1) >18 years old; 2) donation after brainstem death donor; 3) White ethnicity of both donor and recipient; 4) receiving twice-daily TAC formula (Prograf®, Astellas Pharma, Warsaw, Poland) in post-transplant immunosuppressive treatment; 5) availability of donor DNA; 6) completeness of the follow-up (patients who were managed at our outpatient transplantation clinic); and 7) written informed consent to participate in the study. Exclusion criteria were: 1) multiorgan transplantation and 2) primary nonfunctioning kidney transplant. The participants, transplanted 1–7 years prior to recruitment and with stable transplant function, were consecutively enrolled during their follow-up visits at our outpatient transplantation clinic between December 2018 and March 2019 until the required sample size was reached. Recipient blood samples were collected during routine check-up visits after obtaining informed consent. Donor deoxynucleic acid (DNA) was obtained from the tissue typing laboratory of the Department of Immunology, where samples were isolated and stored at −20°C after routine crossmatching before transplantation. Demographic data of the RTRs and their corresponding donors, drug doses, TAC C0 results, and clinical outcomes of transplantation were obtained from medical records. Donors were evaluated using the non-scaled US Kidney Donor Risk Index (KDRI) [22]. No deaths were noted in the observed population, and no patients were lost to follow-up.

The analyzed subjects received triple immunosuppressive therapy consisting of:

In those patients who had positive panel reactive antibody test (PRA) or underwent re-transplantation, the induction therapy was implemented: either anti-thymocyte globulin (Thymoglobulin) or basiliximab. The immunosuppressive regimen was conducted in line with the Polish Transplantation Society guidelines [23].

Whole-blood C0 TAC concentrations were assayed using the chemiluminescence microparticle method and measured routinely during the post-transplant check-ups. This study conforms to the STREGA statement [24].

GENOTYPING:

Real-time polymerase chain reaction (RT-PCR) was used to determine the genotype of CYP3A5 (rs776746; g.99672916C>T) single-nucleotide polymorphism (SNP) in deceased donors and RTRs. RTR DNA was extracted from whole blood using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany). The quality and quantity of the DNA samples were verified by electrophoresis using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Analysis was performed using the rs776746 TaqMan® SNP Genotyping Assay (Thermo Fisher Scientific, Vilnius, Lithuania), according to the qPCR protocol for SNP Genotyping (Thermo Fisher Scientific, Carlsbad, US) [25]. RT-PCR was performed using the QuantStudio™ 12 K Flex Real-Time PCR System (Thermo Fisher Scientific, Carlsbad, US) and repeated 3 times to minimize any genotyping errors. Data analysis was performed using TaqMan® Genotyper software (Thermo Fisher Scientific).

OUTCOME MEASURES:

We compared subgroups defined by (1) donor and (2) recipient CYP3A5 expressor status. The primary outcome was graft loss observed within 5 post-transplant years. It was defined as a loss of renal function resulting in the need for long-term dialysis, graftectomy, or re-transplantation [26]. Secondary outcomes included:

ETHICS:

The study protocol was reviewed and approved by the institutional review board of the Medical University of Warsaw (IRB protocol number: KB/203/2018). This study conforms to the Declaration of Helsinki, Council for International Organizations of Medical Sciences Guidelines, and International Conference on Harmonization of Good Clinical Practice.

STATISTICAL ANALYSIS:

Continuous variables were tested for normality using the Shapiro-Wilk test along with the Q-Q plot analysis and reported as mean (M) and standard deviation (SD) or median (Mdn) with the first and third quartile (interquartile range, IQR), as appropriate. Categorical data were reported as the number of events and percentages. Hardy-Weinberg equilibrium and the chi-squared test were used to evaluate the allele frequencies. Levene’s test was used to assess the equality of variances. Differences between subgroups were tested using the t test, Welch’s t test, or Mann-Whitney U test for continuous variables and the chi-squared or Fisher’s exact test for categorical variables. The primary outcome was evaluated using Kaplan-Meier curves, log-rank test, and Cox proportional hazards analysis. Logistic regression was used to estimate secondary endpoints. All multivariable models were created using backward elimination. Potential risk factors were selected based on a literature search [2,3,28,31,32]. Owing to the small sample size, we could only include a limited number of covariates in our models. Therefore, in some cases, 2 alternative models were created. Missing data were deleted pairwise. Statistical significance was defined as a 2-sided P value <.05. All tests and plots were created using the survival, survminer, transplantr, and CoxR2 packages in R 4.1.2 (R Core Team, 2021).

Results

GENOTYPING, DONOR, AND PATIENT CHARACTERISTICS:

The 95 recipients received kidneys from 63 donors. The call rate of the tested samples from both donors and recipients was 100% (n=158). CYP3A5 *1/*3 (TC) was identified in 22 (13.9%) and *3/*3 (CC) in 136 (86.1%) individuals. No wild-type homozygotes *1/*1 (TT) were found in the tested samples. The genotype distribution was consistent with the HWE χ2 (2, 158)=0.9, P=.642. The allele frequencies were T=0.070 and C=0.930, which is consistent with the data from the 1000 Genomes Project [5] for the European population (T=0.057 and C=0.943). The CYP3A5 genotypes were divided into 2 subgroups: expressors (CYP3A5 *1/*3) and non-expressors (CYP3A5 *3/*3). There were 8 (12.7%) and 14 (14.7%) CYP3A5 expressors among donors and recipients, respectively.

RTRs were divided into subgroups according to the presence or absence of CYP3A5 coding alleles in 1) donors (D+, donor expressor; D−, donor non-expressor) and 2) recipients (R+, recipient expressor; R−, recipient non-expressor). The demographic and clinical characteristics are provided in Table 1.

ASSOCIATION OF POLYMORPHISMS WITH TAC EXPOSURE:

The median TAC C0 levels according to CYP3A5 expression in both recipients and donors within 3 years of follow-up are shown in Supplementary Figure 1.

In recipients who received kidneys from CYP3A5 expressors, TAC C₀ was significantly lower on the third day after transplantation than in non-expressors (Mdn [IQR], 6.0 [4.5–6.7] ng/mL vs 10.9 [6.8–16.5] ng/mL, respectively; U = 248.50, P<.001). In RTRs, carrying the CYP3A5 *1/*3 genotype also resulted in lower TAC C₀ concentrations on the third (Mdn [IQR], 6.3 [4.9–7.6] ng/mL vs 10.9 [6.5–16.4] ng/mL, U=281.50, P=.003), and fifth post-transplant days (Mdn [IQR], 7.5 [6.2–8.9] ng/mL vs 10.7 [8.4–14.8] ng/mL; U=151.50, P=.009). No such correlation was observed during further follow-up when the drug doses were TAC C₀-adjusted.

ASSOCIATION BETWEEN POLYMORPHISMS AND TRANSPLANTATION OUTCOMES:

The parameters considered relevant for transplantation outcomes, such as graft loss, BPAR, proteinuria, and DGF, were assessed in CYP3A5 expressing grafts and recipients, and are summarized in Table 2 and Supplementary Figure 2.

GRAFT LOSS: We conducted survival analysis to evaluate the occurrence of graft loss in predefined subgroups (Figure 1). The number of events was 8 (8.4%) within the 5-year follow-up. We found that RTRs with grafts from CYP3A5 expressors were at greater risk of graft loss (Figure 1A), log-rank test, χ2 (1)=10.8, P<.001. Cox proportional hazard analysis revealed donor CYP3A5 expressor status to be a risk factor for graft loss when adjusted for unscaled US KDRI (adjusted hazard ratio, 95% confidence interval [CI]; 10.61 (2.28–49.42), P=.003, Table 3). We also created a Cox model adjusted for BPAR episodes, which also significantly increased the risk of loss of graft function; however, the model was overfitted due to the small number of observations (Supplementary Table 1).

SECONDARY OUTCOMES:

BPAR episodes occurred in 12 patients (12.6%) within the first postoperative year. Logistic regression analysis showed that donor CYP3A5 expression status increased the risk of BPAR from the 2^nd to the 12^th post-transplant month, which occurred in 31.3% of kidneys carrying the CYP3A5*1 allele vs 7.6% of others (adjusted odds ratio [OR], 95% CI; 4.36, 1.08–17.6; P=.038). No difference was observed within the 1^st month and in total within the 1^st year postoperatively.

Donor CYP3A5 expressor status also increased the risk of proteinuria within the first post-transplant year (adjusted OR, 95%CI: 5.90, 1.40–24.82; P=.015, Table 4) and after that time, within the study follow-up (adjusted OR, 95%CI; 4.49, 1.28–15.72; P=.019). No differences were detected in proteinuria within the first post-transplant month and in delayed graft function. Owing to the small sample size and number of events, analysis of other endpoints was unfeasible. No significant associations were detected regarding recipient CYP3A5 expressor status.

The function of the allogeneic kidney was evaluated using eGFR during 3 years of follow-up. No significant difference was observed, regardless of donor CYP3A5 expression, until the third post-transplant year, when recipient CYP3A5 expression was associated with lower eGFR (M [SD], 59.55 [21.01] ml/min/1.73 m2 vs 45.46 [16.73] ml/min/1.73 m2; t[87]=−2.37, P=.020) (Supplementary Table 2).

Discussion

LIMITATIONS:

This study has several limitations, among which the most important are its retrospective design and the small number of patients included in the analyses. As the funding was limited, we were only able to include a limited number of patients, which might have the same impact, mainly on the secondary analyses. Moreover, in our center, no protocol biopsies are routinely performed, resulting in possible underestimation of BPAR occurrence. We are aware that the method of choice in TAC C₀ determination is LC-MS/MS, and the use of immunoenzymatic assay may provide a measurement error, but it was not available in our center when the data were collected. Finally, the results of our study cannot be extrapolated to other populations owing to the different prevalence of the evaluated polymorphisms worldwide.

Conclusions

In conclusion, our data showed that the donor CYP3A5 genotype is a risk factor for graft loss and other inferior transplantation outcomes (BPAR and proteinuria) in the White population. Therapeutic drug monitoring does not appear to help prevent this phenomenon. To explain this observation, new insights into the mechanisms underlying local TAC metabolism are required. It is a potential new drug target for improving transplant outcomes by individualizing immunosuppressive therapy. Prospective studies are required to evaluate the clinical relevance of our findings. Considering this evidence, we suggest that pharmacogenetic testing, particularly CYP3A5 genotyping, should be considered before renal transplantation to facilitate a personalized patient approach.