The Influence of Tacrolimus Exposure and Metabolism on the Outcomes of Kidney Transplants
Abstract
:1. Introduction
2. Materials and Methods
2.1. Study Design and Patients
2.2. Biomarker Measurement
2.3. Histological Analysis
2.4. Immunosuppression and Tacrolimus Monitoring
2.5. Statistical Analysis
3. Results
3.1. Characteristics of the Study Population
3.2. Graft Biopsy Group Analysis
3.3. Association between Tacrolimus Monitoring and Biomarkers
3.4. Association between Tacrolimus Monitoring and Rejection
3.5. Tacrolimus Measurements and Graft Function
4. Discussion
5. Conclusions
- -
- The data in this study do not show an association between TAC exposure/metabolism and IF/TA progression during the first year after kidney transplantation. However, a higher TAC CV tertile was associated with a higher chronicity score at a one-year biopsy.
- -
- Faster TAC metabolism was associated with reduced kidney graft function and an increased risk of rejection. Calculating the C/D ratio at three and six months after transplantation may help to identify patients at risk for acute rejection and deterioration of graft function and be a simple and inexpensive tool that is useful for physicians in their daily clinical practice. Based on this finding, we recommend considering more frequently monitoring fast tacrolimus metabolizers and a more cautious tapering of other immunosuppressive medicaments.
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- Measuring tacrolimus blood level variability over six months to one year may help to identify patients at a greater risk of progression for chronic graft lesions and reduced long-term graft function. It may also point to potential non-adherence to immunosuppressive treatment.
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- uNGAL, a possible marker of CNI-induced renal injury, negatively correlated with TAC C0 and C/D ratio at three months and one year and with IF/TA and chronicity scores at three-month biopsies.
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
BMI | body mass index |
C/D | concentration doses ratio |
CI | confidence interval |
DGF | delayed graft function |
ECD | expanded-criteria donor |
eGFR | estimated glomerular filtration rate |
HD | hemodialysis |
IF/TA | interstitial fibrosis and tubular atrophy |
KIM-1 | kidney injury molecule-1 |
KRT | kidney replacement therapy |
NGAL | neutrophil gelatinase-associated lipocalin |
MMF | mycophenolate mofetil |
OR | odds ratio |
TAC CV | tacrolimus coefficient of variation |
TAC C0 | tacrolimus trough concentration. |
References
- Organ Procurement and Transplantation Network, United Network for Organ Sharing/Scientific Registry of Transplant; Recipients. Available online: https://srtr.transplant.hrsa.gov/annual_reports/2022/Kidney.aspx (accessed on 10 April 2024).
- Andrews, L.M.; Li, Y.; De Winter, B.C.M.; Shi, Y.-Y.; Baan, C.C.; Van Gelder, T.; Hesselink, D.A. Pharmacokinetic Considerations Related to Therapeutic Drug Monitoring of Tacrolimus in Kidney Transplant Patients. Expert Opin. Drug Metab. Toxicol. 2017, 13, 1225–1236. [Google Scholar] [CrossRef] [PubMed]
- Schütte-Nütgen, K.; Thölking, G.; Steinke, J.; Pavenstädt, H.; Schmidt, R.; Suwelack, B.; Reuter, S. Reuter Fast Tac Metabolizers at Risk—It Is Time for a C/D Ratio Calculation. J. Clin. Med. 2019, 8, 587. [Google Scholar] [CrossRef] [PubMed]
- Kwiatkowska, E.; Kwiatkowski, S.; Wahler, F.; Gryczman, M.; Domańki, L.; Marchelk-Myśliwiec, M.; Ciechanowski, K.; Drozd-Dabrowska, M. C/D Ratio in Long-Term Renal Function. Transplant. Proc. 2019, 51, 3265–3270. [Google Scholar] [CrossRef] [PubMed]
- Jouve, T.; Fonrose, X.; Noble, J.; Janbon, B.; Fiard, G.; Malvezzi, P.; Stanke-Labesque, F.; Rostaing, L. The TOMATO Study (Tacrolimus Metabolization in Kidney Transplantation): Impact of the Concentration–Dose Ratio on Death-Censored Graft Survival. Transplantation 2020, 104, 1263–1271. [Google Scholar] [CrossRef] [PubMed]
- Kwiatkowska, E.; Ciechanowski, K.; Domański, L.; Dziedziejko, V.; Przybyciński, J.; Pawlik, A. Intrapatient Variability (IPV) and the Blood Concentration Normalized by the Dose (C/D Ratio) of Tacrolimus—Their Correlations and Effects on Long-Term Renal Allograft Function. Biomedicines 2022, 10, 2860. [Google Scholar] [CrossRef] [PubMed]
- Kim, E.J.; Kim, S.J.; Huh, K.H.; Kim, B.S.; Kim, M.S.; Kim, S.I.; Kim, Y.S.; Lee, J. Clinical Significance of Tacrolimus Intra-Patient Variability on Kidney Transplant Outcomes According to Pre-Transplant Immunological Risk. Sci. Rep. 2021, 11, 12114. [Google Scholar] [CrossRef] [PubMed]
- Leino, A.D.; King, E.C.; Jiang, W.; Vinks, A.A.; Klawitter, J.; Christians, U.; Woodle, E.S.; Alloway, R.R.; Rohan, J.M. Assessment of Tacrolimus Intrapatient Variability in Stable Adherent Transplant Recipients: Establishing Baseline Values. Am. J. Transplant. 2019, 19, 1410–1420. [Google Scholar] [CrossRef] [PubMed]
- Egeland, E.J.; Reisaeter, A.V.; Robertsen, I.; Midtvedt, K.; Strøm, E.H.; Holdaas, H.; Hartmann, A.; Åsberg, A. High Tacrolimus Clearance—A Risk Factor for Development of Interstitial Fibrosis and Tubular Atrophy in the Transplanted Kidney: A Retrospective Single-Center Cohort Study. Transpl. Int. 2019, 32, 257–269. [Google Scholar] [CrossRef] [PubMed]
- Davis, S.; Gralla, J.; Klem, P.; Stites, E.; Wiseman, A.; Cooper, J.E. Tacrolimus Intrapatient Variability, Time in Therapeutic Range, and Risk of De Novo Donor–Specific Antibodies. Transplantation 2020, 104, 881–887. [Google Scholar] [CrossRef] [PubMed]
- Kuypers, D.R.J. Intrapatient Variability of Tacrolimus Exposure in Solid Organ Transplantation: A Novel Marker for Clinical Outcome. Clin. Pharmacol. Ther. 2020, 107, 347–358. [Google Scholar] [CrossRef] [PubMed]
- Brunet, M.; Van Gelder, T.; Åsberg, A.; Haufroid, V.; Hesselink, D.A.; Langman, L.; Lemaitre, F.; Marquet, P.; Seger, C.; Shipkova, M.; et al. Therapeutic Drug Monitoring of Tacrolimus-Personalized Therapy: Second Consensus Report. Ther. Drug Monit. 2019, 41, 261–307. [Google Scholar] [CrossRef] [PubMed]
- Radzevičienė, A.; Marquet, P.; Maslauskienė, R.; Vaičiūnienė, R.; Kaduševičius, E.; Stankevičius, E. Analyses of AUC(0–12) and C0 Compliances within Therapeutic Ranges in Kidney Recipients Receiving Cyclosporine or Tacrolimus. J. Clin. Med. 2020, 9, 3903. [Google Scholar] [CrossRef] [PubMed]
- Lemaitre, F.; Vethe, N.T.; D’Avolio, A.; Tron, C.; Robertsen, I.; De Winter, B.; Denicolo, A.; Koch, B.C.P.; Venkataramanan, R.; Van Gelder, T.; et al. Measuring Intracellular Concentrations of Calcineurin Inhibitors: Expert Consensus from the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Expert Panel. Ther. Drug Monit. 2020, 42, 665–670. [Google Scholar] [CrossRef] [PubMed]
- Sallustio, B.C. Monitoring Intra-Cellular Tacrolimus Concentrations in Solid Organ Transplantation: Use of Peripheral Blood Mononuclear Cells and Graft Biopsy Tissue. Front. Pharmacol. 2021, 12, 733285. [Google Scholar] [CrossRef] [PubMed]
- Redondo-Pachón, D.; Calatayud, E.; Buxeda, A.; Pérez-Sáez, M.J.; Arias-Cabrales, C.; Gimeno, J.; Burballa, C.; Mir, M.; Llinàs-Mallol, L.; Outon, S.; et al. Evolution of Kidney Allograft Loss Causes over 40 Years (1979–2019). Nefrologia 2023, 43, 316–327. [Google Scholar] [CrossRef] [PubMed]
- Naesens, M.; Lerut, E.; Damme, B.V.; Vanrenterghem, Y.; Kuypers, D.R.J. Tacrolimus Exposure and Evolution of Renal Allograft Histology in the First Year After Transplantation. Am. J. Transplant. 2007, 7, 2114–2123. [Google Scholar] [CrossRef] [PubMed]
- Torres, I.B.; Reisaeter, A.V.; Moreso, F.; Âsberg, A.; Vidal, M.; Garcia-Carro, C.; Midtvedt, K.; Reinholt, F.P.; Scott, H.; Castellà, E.; et al. Tacrolimus and Mycophenolate Regimen and Subclinical Tubulo-Interstitial Inflammation in Low Immunological Risk Renal Transplants. Transpl. Int. 2017, 30, 1119–1131. [Google Scholar] [CrossRef] [PubMed]
- Gatault, P.; Kamar, N.; Büchler, M.; Colosio, C.; Bertrand, D.; Durrbach, A.; Albano, L.; Rivalan, J.; Le Meur, Y.; Essig, M.; et al. Reduction of Extended-Release Tacrolimus Dose in Low-Immunological-Risk Kidney Transplant Recipients Increases Risk of Rejection and Appearance of Donor-Specific Antibodies: A Randomized Study. Am. J. Transplant. 2017, 17, 1370–1379. [Google Scholar] [CrossRef]
- Mo, H.; Kim, S.-Y.; Min, S.; Han, A.; Ahn, S.; Min, S.-K.; Lee, H.; Ahn, C.; Kim, Y.; Ha, J. Association of Intrapatient Variability of Tacrolimus Concentration With Early Deterioration of Chronic Histologic Lesions in Kidney Transplantation. Transplant. Direct 2019, 5, e455. [Google Scholar] [CrossRef] [PubMed]
- Vanhove, T.; Vermeulen, T.; Annaert, P.; Lerut, E.; Kuypers, D.R.J. High Intrapatient Variability of Tacrolimus Concentrations Predicts Accelerated Progression of Chronic Histologic Lesions in Renal Recipients. Am. J. Transplant. 2016, 16, 2954–2963. [Google Scholar] [CrossRef] [PubMed]
- Gonzales, H.M.; McGillicuddy, J.W.; Rohan, V.; Chandler, J.L.; Nadig, S.N.; Dubay, D.A.; Taber, D.J. A Comprehensive Review of the Impact of Tacrolimus Intrapatient Variability on Clinical Outcomes in Kidney Transplantation. Am. J. Transplant. 2020, 20, 1969–1983. [Google Scholar] [CrossRef] [PubMed]
- Fu, R.; Tajima, S.; Suetsugu, K.; Watanabe, H.; Egashira, N.; Masuda, S. Biomarkers for Individualized Dosage Adjustments in Immunosuppressive Therapy Using Calcineurin Inhibitors after Organ Transplantation. Acta Pharmacol. Sin. 2019, 40, 151–159. [Google Scholar] [CrossRef]
- Cantaluppi, V.; Dellepiane, S.; Tamagnone, M.; Medica, D.; Figliolini, F.; Messina, M.; Manzione, A.M.; Gai, M.; Tognarelli, G.; Ranghino, A.; et al. Neutrophil Gelatinase Associated Lipocalin Is an Early and Accurate Biomarker of Graft Function and Tissue Regeneration in Kidney Transplantation from Extended Criteria Donors. PLoS ONE 2015, 10, e0129279. [Google Scholar] [CrossRef] [PubMed]
- Port, F.K.; Bragg-Gresham, J.L.; Metzger, R.A.; Dykstra, D.M.; Gillespie, B.W.; Young, E.W.; Delmonico, F.L.; Wynn, J.J.; Merion, R.M.; Wolfe, R.A.; et al. Donor Characteristics Associated with Reduced Graft Survival: An Approach to Expanding the Pool of Kidney Donors1: Transplantation 2002, 74, 1281–1286. 74. [CrossRef]
- Maslauskiene, R.; Vaiciuniene, R.; Tretjakovs, P.; Gersone, G.; Radzeviciene, A.; Bura, A.; Stankevicius, E.; Bumblyte, I.A. Deceased Kidney Donor Biomarkers: Relationship between Delayed Kidney Function and Graft Function Three Years after Transplantation. Diagnostics 2024, 14, 717. [Google Scholar] [CrossRef] [PubMed]
- Loupy, A.; Mengel, M.; Haas, M. Thirty Years of the International Banff Classification for Allograft Pathology: The Past, Present, and Future of Kidney Transplant Diagnostics. Kidney Int. 2022, 101, 678–691. [Google Scholar] [CrossRef] [PubMed]
- Vaulet, T.; Divard, G.; Thaunat, O.; Koshy, P.; Lerut, E.; Senev, A.; Aubert, O.; Van Loon, E.; Callemeyn, J.; Emonds, M.-P.; et al. Data-Driven Chronic Allograft Phenotypes: A Novel and Validated Complement for Histologic Assessment of Kidney Transplant Biopsies. J. Am. Soc. Nephrol. 2022, 33, 2026–2039. [Google Scholar] [CrossRef] [PubMed]
- Kim, H.; Han, A.; Ahn, S.; Min, S.-K.; Ha, J.; Min, S. Association of High Intra-Patient Variability in Tacrolimus Exposure with Calcineurin Inhibitor Nephrotoxicity in Kidney Transplantation. Sci. Rep. 2023, 13, 16502. [Google Scholar] [CrossRef] [PubMed]
- Bonnard, B.; Ibarrola, J.; Lima-Posada, I.; Fernández-Celis, A.; Durand, M.; Genty, M.; Lopez-Andrés, N.; Jaisser, F. Neutrophil Gelatinase-Associated Lipocalin From Macrophages Plays a Critical Role in Renal Fibrosis Via the CCL5 (Chemokine Ligand 5)-Th2 Cells-IL4 (Interleukin 4) Pathway. Hypertension 2022, 79, 352–364. [Google Scholar] [CrossRef]
- Moriya, H.; Mochida, Y.; Ishioka, K.; Oka, M.; Maesato, K.; Hidaka, S.; Ohtake, T.; Kobayashi, S. Plasma Neutrophil Gelatinase-Associated Lipocalin (NGAL) Is an Indicator of Interstitial Damage and a Predictor of Kidney Function Worsening of Chronic Kidney Disease in the Early Stage: A Pilot Study. Clin. Exp. Nephrol. 2017, 21, 1053–1059. [Google Scholar] [CrossRef] [PubMed]
- Chancharoenthana, W.; Leelahavanichkul, A.; Wattanatorn, S.; Avihingsanon, Y.; Praditpornsilpa, K.; Eiam-Ong, S.; Townamchai, N. Alteration of Urinary Neutrophil Gelatinase–Associated Lipocalin as a Predictor of Tacrolimus-Induced Chronic Renal Allograft Fibrosis in Tacrolimus Dose Adjustments Following Kidney Transplantation. PLoS ONE 2018, 13, e0209708. [Google Scholar] [CrossRef]
- Nogare, A.L.; Veronese, F.V.; Carpio, V.N.; Montenegro, R.M.; Pedroso, J.A.; Pegas, K.L.; Gonçalves, L.F.; Manfro, R.C. Kidney Injury Molecule-1 Expression in Human Kidney Transplants with Interstitial Fibrosis and Tubular Atrophy. BMC Nephrol. 2015, 16, 19. [Google Scholar] [CrossRef] [PubMed]
- Malyszko, J.; Koc-Zorawska, E.; Malyszko, J.S.; Mysliwiec, M. Kidney Injury Molecule-1 Correlates With Kidney Function in Renal Allograft Recipients. Transplant. Proc. 2010, 42, 3957–3959. [Google Scholar] [CrossRef]
- Van Timmeren, M.M.; Vaidya, V.S.; Van Ree, R.M.; Oterdoom, L.H.; De Vries, A.P.J.; Gans, R.O.B.; Van Goor, H.; Stegeman, C.A.; Bonventre, J.V.; Bakker, S.J.L. High Urinary Excretion of Kidney Injury Molecule-1 Is an Independent Predictor of Graft Loss in Renal Transplant Recipients. Transplantation 2007, 84, 1625–1630. [Google Scholar] [CrossRef] [PubMed]
- Kremer, D.; Post, A.; Gomes-Neto, A.W.; Groothof, D.; Kunutsor, S.K.; Nilsen, T.; Hidden, C.; Sundrehagen, E.; Eisenga, M.F.; Navis, G.; et al. Plasma Neutrophil Gelatinase-Associated Lipocalin and Kidney Graft Outcome. Clin. Kidney J. 2022, 15, 235–243. [Google Scholar] [CrossRef] [PubMed]
- Bartlett, F.E.; Carthon, C.E.; Hagopian, J.C.; Horwedel, T.A.; January, S.E.; Malone, A. Tacrolimus Concentration-to-Dose Ratios in Kidney Transplant Recipients and Relationship to Clinical Outcomes. Pharmacotherapy 2019, 39, 827–836. [Google Scholar] [CrossRef] [PubMed]
Characteristics | Values |
---|---|
Donor age, years | 51.39 ± 15.92 |
Donor type, SCD/ECD, (%) | 87 (50.0)/87 (50.0) |
Donor gender, male/female, (%) | 97 (55.7)/77 (44.3) |
Donor hypertension, % | 52.9 |
Donor creatinine, µmol/L | 81 (32–332) |
Cold ischemia time, hours * | 15.3 (9.0–34.3) |
Recipient age, years | 49.09 ± 12.88 |
Recipient gender male/female, (%) | 104 (59.8)/70 (40.2) |
Recipient hypertension, % | 82.8 |
Diabetes mellitus, % | 10.9 |
Recipient BMI, kg/m2 | 25.48 (18.2–38.9) |
HLA mismatch * | 3 (1–6) |
Immunological risk, low/medium/high, (%) | 7.0/80.2/12.8 |
DGF, % | 26.4 |
Rejection during first year, % | 13.8 |
Variable | No IF/TA Progression n = 59 | IF/TA Progression n = 72 | p Value |
---|---|---|---|
TAC dose/weight at 3 months (mg/kg) | 0.121 ± 0.044 | 0.129 ± 0.48 | 0.453 |
TAC dose/weight at 1 year (mg/kg) | 0.079 ± 0.046 | 0.085 ± 0.052 | 0.507 |
TAC C0 at 3 months (ng/mL) | 9.14 ± 2.89 | 9.49 ± 2.96 | 0.49 |
TAC C0 at 6 months (ng/mL) | 8.03 ± 2.4 | 7.59 ± 2.5 | 0.33 |
TAC C0 at 12 months (ng/mL) | 7.53 ± 2.32 | 7.56 ± 2.48 | 0.945 |
C/D at 3 months (ng/mL/mg) | 1.18 (0.32–5.73) | 1.09 (0.28–5) | 0.963 |
C/D at 6 months (ng/mL/mg) | 1.28 (0.45–5.7) | 1.33 (0.3–7.5) | 0.235 |
C/D at 12 months (ng/mL/mg) | 1.3 (0.52–4.7) | 1.24 (0.19–5.95) | 0.454 |
Fast metabolizer percentage at 6 months | 46.3 | 39.7 | 0.457 |
Fast metabolizer percentage at 1 year | 40.9 | 30.2 | 0.266 |
TAC-C0 CV, % | 26.27 ± 11.49 | 27.88 ± 10.44 | 0.423 |
Third TAC-C0 tertile CV | 40.07 ± 8.5 | 38.8 ± 6.6 | 0.678 |
Univariate | Multivariate | |||||
---|---|---|---|---|---|---|
OR | 95% CI | p Value | OR | 95% CI | p Value | |
DGF | 4.19 | 1.719–10.214 | 0.002 | 3.935 | 1.329–11.646 | 0.013 |
Recipient age, years | 0.974 | 0.942–1.007 | 0.122 | |||
Diabetes | 3.513 | 1.187–10.396 | 0.023 | 3.882 | 1.150–13.112 | 0.029 |
Donor age, years | 1.025 | 0.993–1.058 | 0.125 | |||
Donor creatinine, µmol/L | 1.007 | 0.999–1.014 | 0.083 | |||
Cold ischemia time, hours | 1.049 | 0.955–1.142 | 0.34 | |||
TAC C0 at 6 months, ng/mL | 0.78 | 0.622–0.977 | 0.031 | 0.752 | 0.570–0.991 | 0.043 |
Fast metabolizer at 3 months | 3.017 | 1.214–7.497 | 0.017 | |||
Fast metabolizer at 6 months | 2.736 | 1.077–6.951 | 0.034 | |||
Fast metabolizer at 1 year | 2.30 | 0.888–5.958 | 0.081 | |||
C/D at 3 months, ng/mL/mg | 0.301 | 0.12–0.752 | 0.01 | 0.301 | 0.110–0.821 | 0.019 |
C/D at 6 months, ng/mL/mg | 0.44 | 0.188–1.049 | 0.064 | |||
C/D at 1 year, ng/mL/mg | 0.444 | 0.205–0.962 | 0.04 |
Univariate | Multivariate | |||||
---|---|---|---|---|---|---|
OR | 95% CI | p Value | OR | 95% CI | p Value | |
Recipient age, years | 1.001 | 0.974–1.029 | 0.933 | |||
Donor age, years | 1.033 | 1.009–1.056 | 0.006 | 1.043 | 1.001–1.086 | 0.045 |
Donor evaluation | 3.175 | 1.484–6.790 | 0.003 | |||
Donor hypertension | 2.498 | 1.199–5.208 | 0.015 | |||
Cold ischemia time, hours | 1.117 | 1.013–1.232 | 0.027 | |||
KRT time before transplantation, months | 0.986 | 0.973–1.00 | 0.050 | |||
TAC C0 at 6 months, ng/mL | 0.832 | 0.707–0.979 | 0.027 | |||
Fast metabolizer at 6 months | 2.437 | 1.107–5.365 | 0.027 | 4.654 | 1.197–18.097 | 0.026 |
Rejection during first year | 4.648 | 1.014–21.306 | 0.048 | |||
IF/TA score at 1 year | 2.185 | 1.212–3.939 | 0.009 | |||
Chronicity score at 1 year | 1.788 | 1.179–2.711 | 0.006 | 1.575 | 1.002–2.473 | 0.049 |
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Maslauskiene, R.; Vaiciuniene, R.; Radzeviciene, A.; Tretjakovs, P.; Gersone, G.; Stankevicius, E.; Bumblyte, I.A. The Influence of Tacrolimus Exposure and Metabolism on the Outcomes of Kidney Transplants. Biomedicines 2024, 12, 1125. https://doi.org/10.3390/biomedicines12051125
Maslauskiene R, Vaiciuniene R, Radzeviciene A, Tretjakovs P, Gersone G, Stankevicius E, Bumblyte IA. The Influence of Tacrolimus Exposure and Metabolism on the Outcomes of Kidney Transplants. Biomedicines. 2024; 12(5):1125. https://doi.org/10.3390/biomedicines12051125
Chicago/Turabian StyleMaslauskiene, Rima, Ruta Vaiciuniene, Aurelija Radzeviciene, Peteris Tretjakovs, Gita Gersone, Edgaras Stankevicius, and Inga Arune Bumblyte. 2024. "The Influence of Tacrolimus Exposure and Metabolism on the Outcomes of Kidney Transplants" Biomedicines 12, no. 5: 1125. https://doi.org/10.3390/biomedicines12051125
APA StyleMaslauskiene, R., Vaiciuniene, R., Radzeviciene, A., Tretjakovs, P., Gersone, G., Stankevicius, E., & Bumblyte, I. A. (2024). The Influence of Tacrolimus Exposure and Metabolism on the Outcomes of Kidney Transplants. Biomedicines, 12(5), 1125. https://doi.org/10.3390/biomedicines12051125