Impact of cytochrome P450 3A5 expression on clinical outcomes in renal transplant recipients receiving tacrolimus-based immunosuppression

Abstract

BACKGROUND

Kidney transplantation is an effective renal replacement therapy for improving survival and quality of life in chronic kidney disease patients. Kidney transplant recipients need lifelong immunosuppression to prevent rejection and allograft dysfunction. Tacrolimus, a calcineurin inhibitor, is metabolized differently based on cytochrome P450 3A (CYP3A)5 genetic variations and this impacts the graft outcome.

AIM

To examine the clinical outcomes in kidney transplant recipients affected by the variable metabolism of tacrolimus due to the CYP3A5 genetic variation, emphasizing personalized immunosuppression strategies to optimize efficacy, minimize toxicity, and enhance long-term graft survival.

METHODS

A retrospective study was conducted at a tertiary care center in Central India on 95 kidney transplant recipients. Patient demographics, medical history, CYP3A5 polymorphism, post-transplant investigations, graft biopsy results, preexisting comorbidities, history of post–kidney transplant infections, and new onset diabetes after transplantation (NODAT) was collected. Tacrolimus was initiated at 0.1 mg/kg/day for CYP3A5 expressors and 0.05 mg/kg/day for non-expressors, with dose adjustments to maintain target C0 levels of 7-10 ng/mL for first 6 months and 5-7 ng/mL from 6 months to 12 months posttransplant. Patients were followed regularly for one year for glomerular filtration rate (GFR), creatinine, and the tacrolimus trough concentration (ng/mL)/daily tacrolimus dose (mg/kg/day) ratio (C/D). A P value ≤ 0.05 was considered statistically significant.

RESULTS

Kidney transplant recipients were classified as expressors (CYP3A51 carriers, n = 35) and non-expressors (CYP3A5*3*3, n = 60). Both groups were comparable for age, sex, and donor characteristics. Tacrolimus dose was comparable post-transplant except at 6 months and 12 months, where expressors required higher doses. Kidney function (creatinine and estimated GFR), NODAT, hypomagnesemia, and infections showed no significant differences between the two groups over 12 months of follow-up. Biopsy-proven acute rejection (BPAR) was found to be more in expressors (22.9% vs 13.3%, P = 0.2340) though it was not found to be statistically significant. Non-expressors had a significantly higher tacrolimus levels and C/D ratio at multiple follow-ups.

CONCLUSION

CYP3A5 expressors require higher tacrolimus doses to maintain therapeutic levels as compared to non-expressors. BPAR was higher in expressors but the difference was not significant. Graft function, infection rate, and NODAT were comparable irrespective of CYP3A5 expression status, emphasizing the importance of pretransplant CYP3A5 genotyping and therapeutic drug monitoring to guide tacrolimus dosing for individualized immunosuppressive management.

Key Words: Cytochrome P450 3A5 expression; Polymorphism; Renal transplant; Tacrolimus; Tacrolimus trough concentration (ng/mL)/daily tacrolimus dose (mg/kg/day) ratio

Core Tip: This study evaluated the effect of cytochrome P450 3A (CYP3A)5 polymorphism on tacrolimus pharmacokinetics and clinical outcomes in renal transplant recipients. CYP3A5 expressors required significantly higher tacrolimus doses than non-expressors from 6 months onward to maintain therapeutic levels. Non-expressors consistently showed higher tacrolimus trough concentration (ng/mL)/daily tacrolimus dose (mg/kg/day) ratios, reflecting slower drug metabolism. Despite these differences, no significant impact was seen on acute rejection rates, renal function, or complications such as new onset diabetes after transplantation and hypomagnesemia. These findings emphasize the importance of genotype-guided tacrolimus dosing for individualized immunosuppressive management, while therapeutic drug monitoring helps mitigate clinical variability, ensuring comparable outcomes between expressors and non-expressors.

INTRODUCTION

The global burden of end-stage kidney disease is on the rise and successful kidney transplantation is the most effective kidney replacement therapy improving quality of life. Lifelong immunosuppressive therapy is required in kidney transplant recipients to prevent both early and late acute rejection episodes and chronic allograft dysfunction. The long-term outcome depends on achieving a balance between preventing rejection and minimizing drug-related toxicity through appropriate immunosuppressive regimens. Tacrolimus is a calcineurin inhibitor which suppresses the T-cell function by inhibiting the synthesis of interleukin-2 and cytokines, thus preventing acute and chronic rejection[1]. Cytochrome P450 3A (CYP3A)5 is the main enzyme for tacrolimus metabolism while there are some other enzymes and proteins with a minor role in its metabolism, including UDP-glucuronosyltransferases and P-glycoprotein. The tacrolimus trough levels with same dose vary significantly due to inter-individual variability in its metabolism by the genetic polymorphisms in the CYP3A5 gene belonging to cytochrome P450 3A enzyme system. Genetic variations in the CYP3A5 gene categorize patients into two metabolizer phenotypes: (1) Expressors (carrying at least one functional CYP3A51 allele); and (2) Non-expressors (homozygous for the non-functional CYP3A53/*3 genotype)[2]. Expressors exhibit higher CYP3A5 activity, leading to faster tacrolimus metabolism and potentially subtherapeutic drug levels if standard doses are administered, increasing the risk of graft rejection. Non-expressors have lower CYP3A5 activity, resulting in slower tacrolimus metabolism and a higher risk of over-immunosuppression and nephrotoxicity, infection, and metabolic complications such as diabetes or dyslipidemia[3].

Tacrolimus is the primary maintenance immunosuppressant used after kidney transplantation with over 95% of recipients on tacrolimus-based regimen and CYP3A5 expression status has a significant impact on pharmacokinetics and clinical outcomes[4]. This study was done to investigate the clinical outcomes between expressors and non-expressors in renal transplant recipients receiving tacrolimus-based immunosuppression. We intend to stress on personalized immunosuppression strategies with optimal efficacy and minimal toxicity leading to improved long-term graft and patient survival.

MATERIALS AND METHODS

Research question

Does CYP3A5 genetic polymorphism influence clinical outcomes in renal transplant recipients receiving tacrolimus-based immunosuppression?

Methods

A retrospective study was performed at a tertiary care center in Central India from 2021 to 2025. Medical records of 95 patients who underwent kidney transplantation with pretransplant CYP3A5 genetic study and receiving a tacrolimus-based primary maintenance immunosuppressive regimen (TAC) were evaluated and included in the study. Kidney recipients aged 18 years and above were included. Patients expired within the first 3 months post-transplantation or lost to follow-up were excluded. Demographic characteristics, previous medical history, human leukocyte antigen compatibility between donor and recipient, panel reactive antibody levels, post-transplant graft function, graft biopsy results (if performed), infections, and new onset diabetes after transplantation (NODAT) were documented.

Polymerase chain reaction-restriction fragment length polymorphism for identifying CYP3A5 polymorphism

Genomic DNA was extracted from whole blood samples utilizing the GeneAll DNA viral kit (GeneAll Biotechnology Co., Seoul, Korea) in accordance with the manufacturer’s protocol. The extracted DNA was subsequently stored at -70 °C until further analysis. Polymerase chain reaction (PCR) amplification was conducted using a Mastercycler (Eppendorf, Hamburg, Germany) with primers previously described in the literature. The thermal cycling conditions consisted of an initial denaturation at 95 °C for 5 minutes, followed by 35 cycles of denaturation at 95 °C for 30 seconds, annealing at 58 °C for 30 seconds, and extension at 72 °C for 30 seconds, with a final extension step at 72 °C for 7 minutes. Restriction enzyme digestion was carried out in a total reaction volume of 50 μL, comprising 1 μL of SspI enzyme, 5 μL of reaction buffer, 34 μL of sterile water, and 10 μL of PCR-amplified product. CYP3A5 genotyping was performed through electrophoresis on a 2% agarose gel stained with ethidium bromide (10 mg/mL) and visualized under ultraviolet illumination. The accuracy of genotyping results was validated through direct sequencing using a 3130 XL Genetic Analyzer (Applied Biosystems, Foster City, CA, United States).

Treatment plan

After CYP3A5 genotyping analysis, TAC was started at a dose of 0.1 mg/kg/day for expressors and at a dose of 0.05 mg/kg/day non-expressors. Subsequent doses were adjusted to achieve target C0 levels of 7-10 ng/mL for first 6 months and 5-7 ng/mL after 6 months. Patients were followed twice weekly during the first month, once weekly during the second month, bimonthly for the subsequent 2–3 months, and monthly thereafter until the completion of the first year. Glomerular filtration rate (GFR) was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation. Tacrolimus concentrations were measured using a chemiluminescent microparticle immunoassay on the Architect i2000 system (Abbott Diagnostics Laboratories, Abbott, IL, United States). Data on tacrolimus dosing and monitoring were collected, including the prescribed dosage regimen, measured whole-blood trough concentrations (C0), and any physician-initiated dose adjustments. These parameters were assessed at baseline, followed by evaluations on days 5, 15, 30, 45, 60, and 120, as well as at months 6, 9, and 12. The tacrolimus trough concentration (ng/mL)/daily tacrolimus dose (mg/kg/day) (C/D) ratio was calculated to assess an individual’s drug metabolism and dose-adjustment requirements.

Statistical analysis

Statistical analyses were conducted using Statistical Package for the Social Sciences version 20.0 (IBM, Armonk, NY, United States) and GraphPad Prism version 8.0. Categorical variables were analyzed using the χ² test or Fisher’s exact test, as appropriate. Continuous variables were compared between two groups using Student’s t-test for normally distributed data and the Mann-Whitney U test for non-normally distributed data. A P value ≤ 0.05 was considered statistically significant. The study was approved by the local institutional ethics committee.

RESULTS

The study population was stratified into two groups based on polymorphism expression: (1) Expressors, including heterozygous and wild-type variants having at least one copy of CYP3A5*1 (n = 35); and (2) Non-expressors, comprising the homozygous variant of CYP3A5*3*3 (n = 60). Gender distribution between expressors (77.1% male) and non-expressors (80% male) was comparable (P = 0.7431). Most recipients were between age group 31-50 years in both groups (48.6% in expressors vs 51.7% in non-expressors) (P = 0.7399). Pre-transplant diabetes mellitus was similar in the two groups (17.1% in expressors and 16.7% in non-expressors) (P = 0.9525). Hypertension was higher in expressors (60.0%) as compared to non-expressors (50.0%), but the difference was not significant (P = 0.3483). Twelve recipients (12.6%) underwent deceased donor transplant and 83 (87.4%) underwent live donor transplant. Mother was the most common donor in both groups, followed by spouse, father, and siblings (Table 1).

Table 1 Clinical characteristics of renal transplant patients between expressor and non-expressors.

Variable	Expressors (n = 35)		Non-expressors (n = 60)		P value
	n	%	n	%
Gender
Male	27	77.1	48	80	0.7431
Female	8	22.9	12	20
Age group
10-30 years	13	37.1	18	30.0	0.7399
31-50 years	17	48.6	31	51.7
> 50 years	5	14.3	11	18.3
Comorbidities
Diabetes	6	17.1	10	16.7	0.9525
Hypertension	21	60.0	30	50.0	0.3483
Donor
Father	2	5.7	3	5.0	0.2172
Mother	8	22.9	16	26.7
Wife	3	8.6	4	6.7
Other	22	62.9	37	61.7
Kidney
Live	30	85.7	53	88.3	0.7123
Cadaveric	5	14.3	7	11.7

The mean tacrolimus dose at different time points showed no statistically significant differences between the two groups except at the 6th month (0.06 mg/kg ± 0.03 mg/kg vs 0.04 mg/kg ± 0.03 mg/kg, P = 0.002) and at the 12^th month (0.06 mg/kg ± 0.01 mg/kg vs 0.04 mg/kg ± 0.03 mg/kg, P = 0.002), where expressors received significantly higher doses (Table 2). Tacrolimus trough levels demonstrated no significant differences during early post-transplant period (up to 120 days), with P values ranging from 0.1180 to 0.8848. However, from 6 months onwards, tacrolimus trough levels were significantly higher in expressors compared to non-expressors. At 6 months, the mean tacrolimus level in expressors was 6.7 ng/mL ± 2.2 ng/mL vs 5.3 ng/mL ± 1.2 ng/mL in non-expressors (P = 0.001). This trend continued at 9 months (6.5 ng/mL ± 1.2 ng/mL vs 4.6 ng/mL ± 1.2 ng/mL, P < 0.001) and at 12 months (6.2 ng/mL ± 1.2 ng/mL vs 4.9 ng/mL ± 0.7 ng/mL, P < 0.001) (Table 3).

Table 2 Association of cytochrome P450 3A5 polymorphism with tacrolimus doses in expressors and non-expressors.

Timeline	Expressors (n = 35) (mean ± SD)	Non-expressors (n = 60) (mean ± SD)	P value
Tacrolimus dose (mg/kg)
At 5 days	0.08 ± 0.14	0.05 ± 0.02	0.1049
At 15 days	0.05 ± 0.03	0.05 ± 0.03	-
At 30 days	0.05 ± 0.03	0.05 ± 0.03	-
At 45 days	0.06 ± 0.03	0.05 ± 0.03	0.1205
At 90 days	0.06 ± 0.03	0.05 ± 0.03	0.1205
At 120 days	0.06 ± 0.03	0.05 ± 0.03	0.1205
At 6 months	0.06 ± 0.03	0.04 ± 0.03	0.002
At 9 months	0.06 ± 0.02	0.05 ± 0.03	0.082
At 12 months	0.06 ± 0.01	0.04 ± 0.03	0.002

Table 3 Association of cytochrome P450 3A5 polymorphism with tacrolimus trough level in expressors and non-expressors.

Timeline	Expressors (n = 35) (mean ± SD)	Non-expressors (n = 60) (mean ± SD)	P value
Tacrolimus trough level (ng/mL)
At 5 days	7.6 ± 3.3	7.7 ± 3.2	0.8848
At 15 days	6.1 ± 3.0	6.8 ± 2.5	0.2249
At 30 days	6.0 ± 2.2	6.4 ± 2.0	0.3672
At 45 days	5.6 ± 2.2	6.2 ± 1.5	0.1180
At 90 days	6.7 ± 2.9	5.9 ± 2.6	0.1690
At 120 days	6.6 ± 1.9	6.1 ± 1.9	0.2191
At 6 months	6.7 ± 2.2	5.3 ± 1.2	0.001
At 9 months	6.5 ± 1.2	4.6 ± 1.2	< 0.001
At 12 months	6.2 ± 1.2	4.9 ± 0.7	< 0.001

The mean creatinine levels demonstrated a declining trend in both groups, with no statistically significant differences observed at 1 month (P = 0.1694), 3 months (P = 0.0838), 6 months (P = 0.2165), and 9 months (P = 0.3958). By 12 months, the creatinine levels were comparable between the two groups (1.7 mg/dL ± 0.5 mg/dL vs 1.7 mg/dL ± 0.4 mg/dL). Similarly, estimated GFR levels showed no statistically significant differences at 1 month (P = 0.1531), 3 months (P = 0.4321), 6 months (P = 0.8988), 9 months (P = 0.3213), or 12 months (P = 0.5573) (Table 4).

Table 4 Renal parameters of patients with respect to polymorphism.

Renal parameter	Expressors (n = 35) (mean ± SD)	Non-expressors (n = 60) (mean ± SD)	P value
Creatinine (mg/dL)
At 1 month	1.8 ± 1.6	1.5 ± 0.4	0.1694
At 3 months	1.9 ± 1.7	1.5 ± 0.4	0.0838
At 6 months	2.0 ± 1.7	1.7 ± 0.6	0.2165
At 9 months	1.9 ± 1.7	1.7 ± 0.5	0.3958
At 12 months	1.7 ± 0.5	1.7 ± 0.4	-
Estimated glomerular filtration rate levels (mL/minute/1.73 m2)
At 1 month	56.4 ± 19.4	62.6 ± 20.7	0.1531
At 3 months	56.2 ± 24.8	59.7 ± 18.2	0.4321
At 6 months	54.2 ± 28.4	54.8 ± 17.5	0.8988
At 9 months	57.9 ± 28.6	53.2 ± 17.4	0.3213
At 12 months	51.0 ± 24.0	53.4 ± 15.7	0.5573

The incidence of NODAT was comparable in expressors and non-expressors (2.9% vs 5.0%, P = 0.6178). Hypomagnesemia was frequently observed among expressors compared to non-expressors but the difference was not significant (62.9% vs 51.7%, P = 0.2920). Post-transplant tuberculosis was seen in 8.6% of expressors and 13.3% of non-expressors (P = 0.8710). Tacrolimus discontinuation and switch to cyclosporine or sirolimus was required in 22.9% of expressors and 16.7% of non-expressors (P = 0.4600). Biopsy-proven acute rejection (BPAR) was higher in expressors (22.9%) compared to non-expressors (13.3%), but the difference was found to be non-significant (P = 0.2340) (Table 5).

Table 5 Complications and outcomes observed in expressor vs non-expressors.

Variable	Expressors (n = 35)		Non-expressors (n = 60)		P value
	n	%	n	%
New onset diabetes after transplantation
Yes	1	2.9	3	5.0	0.6178
No	34	97.1	57	95.0
Hypomagnesemia
Yes	22	62.9	31	51.7	0.2920
No	13	37.1	29	48.3
Tuberculosis after transplant
Yes	3	8.6	8	13.3	0.8710
No	32	91.4	52	86.7
Tacrolimus discontinued permanently
Yes	8	22.9	10	16.7	0.4600
No	27	77.1	50	83.3
Biopsy-proven acute rejection
Yes	8	22.9	8	13.3	0.2340
No	27	77.1	52	86.7

The C/D ratio varied significantly between expressors and non-expressors over time. Non-expressors exhibited a significantly higher C/D ratio at multiple follow-ups, particularly at 15 days (P = 0.0425), 30 days (P = 0.0348), 45 days (P = 0.0018), 120 days (P = 0.0016), and 6 months (P = 0.0158) (Figure 1).

Figure 1 Tacrolimus trough concentration (ng/mL)/daily tacrolimus dose (mg/kg/day) ratio in expressors vs non expressors. C/D: Tacrolimus trough concentration (ng/mL)/daily tacrolimus dose (mg/kg/day).

DISCUSSION

This study evaluated the impact of CYP3A5 expression on clinical outcomes in renal transplant recipients receiving tacrolimus-based immunosuppression. We observed a significant difference in tacrolimus dose requirements between the two groups. At 30 days post-transplant, tacrolimus dosing was comparable between the groups, with both expressors and non-expressors receiving a dose of 0.05 mg/kg ± 0.03 mg/kg. However, from day 45 to day 120, the expressors group required higher doses (0.06 mg/kg ± 0.03 mg/kg) compared to the non-expressors group (0.05 mg/kg ± 0.03 mg/kg), though the difference was not statistically significant (P = 0.1205). By the sixth month, the expressors group required significantly higher doses (0.06 mg/kg ± 0.03 mg/kg) than the non-expressors group (0.04 mg/kg ± 0.03 mg/kg, P = 0.002). This trend continued beyond 6 months, with the expressors group consistently requiring higher doses to maintain therapeutic drug levels. The dose difference remained statistically significant, with expressors requiring 0.06 mg/kg ± 0.03 mg/kg vs 0.04 mg/kg ± 0.03 mg/kg at 6 months (P = 0.002) and 0.06 mg/kg ± 0.01 mg/kg vs 0.04 ± 0.03 mg/kg at 12 months (P = 0.002). These findings suggest that expressors exhibit a higher metabolic clearance of tacrolimus, necessitating dose escalation to maintain therapeutic drug levels, whereas non-expressors demonstrate a more stable pharmacokinetic profile with lower dose requirements. The observed differences highlight the importance of genotype-guided immunosuppressive strategies to optimize tacrolimus dosing, ensuring adequate immunosuppression while minimizing the risk of rejection in expressors and drug-related toxicity in non-expressors. These findings are similar to a study conducted in 165 renal transplant recipients, which demonstrated that tacrolimus trough concentrations were significantly higher in the CYP3A5 non-expressor group compared to the CYP3A5 expressor group (P < 0.05)[5]. Similar studies conducted in Korean patients carrying the CYP3A5 allele have demonstrated significantly elevated tacrolimus concentrations in homozygous individuals compared to those with at least one CYP3A51 allele. CYP3A5 polymorphism has been shown to significantly influence the attainment of target tacrolimus trough levels[6,7]. Carriers of at least one active allele (CYP3A51) required significantly higher tacrolimus doses compared to CYP3A5*3 homozygotes (CYP3A5 non-expressors)[2].

Conventionally, CYP3A5 expressors and non-expressors are administered tacrolimus at doses 0.15–0.2 mg/kg/day and 0.1 mg/kg/day, respectively. Our study patients received a lower tacrolimus dosage (0.1 mg/kg/day and 0.06-0.08 mg/kg/day in expressors and non expressors, respectively) in accordance to the available data in Indian patients[8]. In our study, non-expressors demonstrated a significantly higher C/D ratio across multiple follow-up assessments compared to expressors. Those observations are similar to previous studies which showed non-expressors with a higher C/D ratio in comparison to expressors[9,10]. CYP3A5 expressors thus metabolize tacrolimus faster and may be at a higher risk of subtherapeutic drug levels, potentially leading to an increased risk of graft rejection. However, our study showed that CYP3A5 polymorphism does not significantly affect clinical outcomes such as acute rejection rates, renal function, or graft survival. Many studies with larger study population have shown no statistically significant association between CYP3A5 polymorphism and BPAR, renal function, graft survival or tacrolimus toxicity[11-13]. A meta-analysis of 1246 kidney transplant recipients found no statistically significant difference in acute rejection rates between CYP3A5 expressers and non-expressors[14]. Those findings suggest that the observed pharmacokinetic differences driven by CYP3A5 expression may not readily translate into clinically significantly different outcomes.

Several factors likely contribute to this apparent dissociation between pharmacokinetics and clinical effects. First, the intensive therapeutic drug monitoring (TDM) and subsequent tacrolimus dose titration implemented in the early post-transplant period likely mitigate the clinical impact of CYP3A5-mediated variations in tacrolimus metabolism. This dynamic dose adjustment allows for rapid attainment and maintenance of target tacrolimus levels, effectively minimizing the clinical consequences of inter-individual pharmacokinetic variability. Second, multiple non-genetic factors, such as steroid doses, serum albumin levels, and drug-drug interactions, also influence tacrolimus levels and may potentially mask the isolated effect of CYP3A5 polymorphisms. The interplay of these factors likely contributes to the overall variability in tacrolimus exposure and may obscure the specific contribution of CYP3A5 genotype to clinical outcomes. In our study, a higher percentage of expressors (22.9%) underwent permanent tacrolimus discontinuation compared to non-expressors (16.7%); however, this difference was not statistically significant (P = 0.4600). Similarly, the choice of alternative immunosuppressants did not differ significantly between the groups (P = 0.4864). Tacrolimus is well-known to contribute to NODAT through mechanisms such as β-cell toxicity and insulin resistance. It also induces magnesium depletion by increasing renal magnesium excretion, a well-documented side effect. However, in our study, neither NODAT nor hypomagnesemia rates differed significantly between the two groups (P = 0.272 and P = 0.3865, respectively). These findings suggest that while CYP3A5 genotype plays a crucial role in tacrolimus pharmacokinetics, its influence on these particular clinical outcomes is limited, likely due to the combined effects of TDM and other clinical variables. Further research exploring the long-term clinical implications of CYP3A5 polymorphism and investigating personalized tacrolimus dosing strategies is warranted.

CONCLUSION

This study highlights the influence of CYP3A5 polymorphism on tacrolimus pharmacokinetics in renal transplant recipients. Demographic and clinical characteristics were largely comparable between expressors and non-expressors, while significant differences were observed in tacrolimus dose requirements and trough levels over time. Expressors required higher doses to achieve therapeutic levels, whereas non-expressors consistently exhibited higher C/D ratios. Despite these pharmacokinetic differences, renal function outcomes, incidence of adverse effects, and post-transplant complications, including new-onset diabetes and acute rejection episodes, did not significantly differ between the groups. These findings underscore the importance of genotype-guided dosing strategies in optimizing tacrolimus therapy while maintaining comparable clinical outcomes across genetic variants. Future studies should explore genotype-guided dosing protocols in larger, multi-center cohorts to validate these findings. Additionally, long-term outcomes and cost-effectiveness of personalized tacrolimus dosing warrant further investigation.