P-glycoprotein, FK-binding Protein-12, and the Intracellular Tacrolimus Concentration in T-lymphocytes and Monocytes of Kidney Transplant Recipients

Abstract

Background . Transplant recipients may develop rejection despite having adequate tacrolimus whole blood predose concentrations (C0). The intra-immune cellular concentration is potentially a better target than C0. However, little is known regarding intracellular tacrolimus concentration in T-lymphocytes and monocytes. We investigated the tacrolimus concentrations in both cell types and their relation with the expression and activity of FK-binding protein (FKBP)-12 and P-glycoprotein (P-gp). Methods . T-lymphocytes and monocytes were isolated from kidney transplant recipients followed by intracellular tacrolimus concentration measurement. FKBP-12 and P-gp were quantified with Western blot, flow cytometry, and the Rhodamine-123 assay. Interleukin-2 and interferon-γ in T-lymphocytes were measured to quantify the effect of tacrolimus. Results . Tacrolimus concentration in T-lymphocytes was lower than in monocytes (15.3 [8.5–33.4] versus 131.0 [73.5–225.1] pg/million cells; P < 0.001). The activity of P-gp (measured by Rhodamine-123 assay) was higher in T-lymphocytes than in monocytes. Flow cytometry demonstrated a higher expression of P-gp (normalized mean fluorescence intensity 1.5 [1.2–1.7] versus 1.2 [1.1–1.4]; P = 0.012) and a lower expression of FKBP-12 (normalized mean fluorescence intensity 1.3 [1.2–1.7] versus 1.5 [1.4–2.0]; P = 0.011) in T-lymphocytes than monocytes. Western blot confirmed these observations. The addition of verapamil, a P-gp inhibitor, resulted in a 2-fold higher intra-T-cell tacrolimus concentration. This was accompanied by a significantly fewer cytokine-producing cells. Conclusions . T-lymphocytes have a higher activity of P-gp and lower concentration of the FKBP-12 compared with monocytes. This explains the relatively lower tacrolimus concentration in T-lymphocytes. The addition of verapamil prevents loss of intracellular tacrolimus during the cell isolation process and is required to ensure adequate intracellular concentration measurement.

Modelling changes in the pharmacokinetics of tacrolimus during pregnancy after kidney transplantation: A retrospective cohort study

“…Lastly, this popPK model is developed for twice‐daily tacrolimus and cannot be extrapolated to once‐daily tacrolimus formulations. To improve this popPK model, further research is required in pregnant kidney transplant recipients by measuring area under the concentration–time curves, and by determining the effect of unbound tacrolimus and intralymphocyte tacrolimus concentrations 54 . Currently, we are setting up such a (nationwide) study.…”

Impact of Infection-Related Immunosuppressant Reduction on Kidney Transplant Outcomes: A Retrospective Study Considering the Temporal Dynamics of Immunosuppressive Requirements

“…Our results suggest that the intensity of immunosuppression and antibiotic prophylaxis significantly influence the frequency and timeline of post-transplant infections. As the pharmacokinetic monitoring alone is insufficient for estimating the intensity of immunosuppression after transplantation, researchers have developed some new techniques such as measuring virus-specific T cell levels in addition to pharmacokinetic monitoring, measuring the viral load of torque teno virus, monitoring the intracellular tacrolimus concentration in T-lymphocytes and other immune cells, et al, to solve this problem [26][27][28]. Optimizing the immunosuppressive protocol based on these new techniques might help us to further reduce the risk of infections and unnecessary ISR in future.…”