Invited critical reviewCalcineurin inhibitors and NFAT-regulated gene expression
Introduction
Calcineurin-inhibitors (CNI) are widely used in organ transplantation and more than 80% of de novo renal and heart transplant recipients are on CNI therapy [1]. Both CNIs, ciclosporin A (CsA) and tacrolimus (Tac), have confirmed an excellent efficacy profile with low acute rejection rates. The current 1-year survival rate of renal allografts reaches 90% [2]. Unexpectedly, the overall renal allograft survival remained stable during the last decades indicating a shift of graft failure due to rejection to consequences of long-term immunosuppression [3]. CNIs have a narrow therapeutic window; therefore, regular monitoring of the drug is necessary to balance sufficient efficacy with minimal toxicity. Common CNI induced side effects include nephrotoxicity, hypertrichosis, gingival overgrowth and hypertension in CsA therapy, and hyperglycemia as well as neurotoxicity in Tac immunosuppression [4], [5], [6], [7]. Most of these adverse reactions are dose dependent. On the other side, sub-therapeutic drug levels significantly increase the risk of allograft rejection. Until now, monitoring of immunosuppressive drugs is performed by pharmacokinetic assessments, mainly by trough concentrations (C0) of the drug. Detailed information about total drug exposure is provided by 12-h pharmacokinetic profiles. The area under the concentration–time curve (AUC0-12) predicts efficacy and safety [8], [9]. However, with 12 blood samples taken in 12 h these pharmacokinetic profiles are very inconvenient, costly and almost not feasible in regular routine practice. Data concerning the relationship between C0 and AUC0-12 are conflicting, whereas C2 levels are reported to correlate most closely with AUC0-12 [9], [10], [11], [12]. There are only a few prospective clinical studies comparing single time point concentrations or limited sampling strategies with the corresponding clinical outcome [9], [12], [13]. Long-term graft loss, acute rejection and nephrotoxicity were associated with deviations in the CsA C2 but not the C0 levels [14], [15]. Therefore C2 monitoring might provide a convenience of a single time point measurement with a good correlation to clinical events. All these methods rely on pharmacokinetic data, which does not reflect the biological effects of CNI on the immune system. In addition, similar drug concentrations cause highly variable pharmacodynamic effects in individual patients, reflecting the heterogeneity of their immunotype.
Altogether, there is a great need for a more informative monitoring beyond the traditional therapeutic drug monitoring by blood concentration. Several approaches have been undertaken to measure the biologic effects of CNI-based immunosuppression including calcineurin phosphatase activity, release of cytokines and gene expression [16], [17], [18], [19], [20], [21].
Section snippets
Mode of action and possible specific pharmacodynamic monitoring assays
Detailed knowledge about the drug-specific mode of action is required to establish and introduce a pharmacodynamic (PD) monitoring assay. The proposed immunosuppressive mode of CsA and Tac is the inhibition of the phosphatase activity of calcineurin after binding of the corresponding CNI–immunophilin complexes [22], [23], Fig. 1. The main substrate of calcineurin in T-cells is the phosphorylated transcription factor NFAT. NFAT dephosphorylation by calcineurin is required for the nuclear
Assessment of CNI therapy by NFAT-regulated gene expression
A PD monitoring assay is only useful if it provides reliable and valid results and if it is easy to perform. Today, gene expression assays are established and used in daily routine. Analysis of gene expression provides reliable and valid results and is used widely in medical diagnostics [32]. Even in PD monitoring assays assessment of gene expression has been used in several studies and concerning several targets [19], [20]. The available technique of real-time PCR provides a fast, highly
NFAT-regulated gene expression: PK/PD studies
An inverse correlation between drug blood concentration and expression of NFAT-regulated genes has been observed in CsA and in Tac treated patients [31], [40]. The highest inhibition of gene expression occurred at the time of the maximum blood concentration 2-h after CsA intake or about 1.5-h after Tac intake. Since the maximal inhibition of NFAT-regulated genes correlates with the peak drug concentration, one might suggest that CsA C2 or Tac C1.5 level reflects immunosuppressive activity of
Clinical studies — efficacy and safety in CsA treated renal allograft recipients
It is obvious that a PD monitoring assay is only useful if a clinical benefit is confirmed. Despite the various methods described for the PD monitoring of CNIs in the literature, until now none of these assays has been implemented in clinical practice. Several clinical studies affirmed the approach of residual NFAT-regulated gene expression as a useful tool with potential to individualize CNI therapy, Table 1.
One of the first prospective observational trials in stable renal allograft recipients
Clinical studies — efficacy and safety in Tac treated renal allograft recipients
The utility and clinical benefit of pharmacodynamic monitoring of Tac therapy by residual NFAT-regulated gene expression was evaluated in stable renal allograft recipients [40]. The median minimal residual gene expression varied widely from 0% to 138% 1.5 h after oral Tac intake. More than half of the 262 stable renal transplant patients exhibited a residual gene expression below 30% and 25 patients exhibited gene expression above 80%. Tac concentration at 1.5 h, but not Tac trough concentration
NFAT-regulated gene expression in other solid organ transplantation
In addition, PD monitoring by residual NFAT-regulated gene expression has been evaluated in other solid organ transplantation like heart and liver allograft recipients with CsA therapy [41], [53]. In the pilot study a strong inhibition of all three NFAT-regulated genes, IL-2, IFNγ and GM-CSF could be shown 2 h after drug intake in kidney, heart and liver transplant patients [31]. Corresponding to the daily CsA dosage heart transplant patients (2.9 (1.4–6.1) mg/kg CsA per day) demonstrated the
NFAT-regulated gene expression in special patient cohorts
In addition, assessment of NFAT-regulated gene expression has been evaluated in pediatrics and senior renal allograft recipients.
In an observational study it could be confirmed that pediatric renal allograft recipients with recurrent infections presented a significantly stronger inhibition of NFAT-regulated gene expression (18.2%) than patients without recurrent infections (31.7%; p = 0.012) [38]. Multivariate regression analysis showed that besides patient age residual NFAT-regulated gene
Conclusions
Several cross-sectional and prospective observational clinical studies in different patient cohorts indicate NFAT-regulated gene expression as promising tool to individualize CsA- and Tac-based immunosuppression. Quantitative analysis of NFAT-regulated gene expression provides a reliable quantification of functional immunosuppression at the target cell levels in solid organ recipients. Especially, beneficial effects concerning adverse events like recurrent infections or malignancies have been
Conflict of interest
None of the authors has any conflicts of interest.
References (62)
- et al.
Immunosuppression: evolution in practice and trends, 1994–2004
Am J Transplant
(2006) - et al.
Lack of improvement in renal allograft survival despite a marked decrease in acute rejection rates over the most recent era
Am J Transplant
(2004) - et al.
Cancer risk in patients on dialysis and after renal transplantation
Lancet
(2000) - et al.
A transient increase of calcineurin phosphatase activity in living-donor kidney transplant recipients with acute rejection
Drug Metab Pharmacokinet
(2010) - et al.
Recognition of NFATp/AP-1 composite elements within genes induced upon the activation of immune cells
J Mol Biol
(1999) - et al.
Spectrophotometric assay for calcineurin activity in leukocytes isolated from human blood
Anal Biochem
(2006) - et al.
Pharmacokinetic and pharmacodynamic correlations of cyclosporine therapy in stable renal transplant patients: evaluation of long-term target C(2)
Int Immunopharmacol
(2003) - et al.
Assessment of immunosuppressive drug interactions: inhibition of lymphocyte function in peripheral human blood
J Immunol Methods
(2003) - et al.
How to do successful gene expression analysis using real-time PCR
Methods
(2010) - et al.
Activity of nuclear factor of activated T cells is independent of the number of peripheral lymphocytes in FTY720-treated patients
Transplant Proc
(2008)
Outcomes of kidney transplantation from older living donors to older recipients
Am J Kidney Dis
Cancer after kidney transplantation in the United States
Am J Transplant
Biomarkers of immunoregulatory status in stable liver transplant recipients undergoing weaning of immunosuppressive therapy
Clin Immunol
Improved graft survival after renal transplantation in the United States, 1988 to 1996
N Engl J Med
Immunosuppressive drugs in kidney transplantation: impact on patient survival, and incidence of cardiovascular disease, malignancy and infection
Drugs
Calcineurin inhibitor nephrotoxicity: longitudinal assessment by protocol histology
Transplantation
Malignancy in renal transplantation
J Am Soc Nephrol
Approaching the therapeutic window for cyclosporine in kidney transplantation: a prospective study
J Am Soc Nephrol
Comparison of trough, 2-hour, and limited AUC blood sampling for monitoring cyclosporin (Neoral) at day 7 post-renal transplantation and incidence of rejection in the first month
Ther Drug Monit
Patient management by Neoral C(2) monitoring: an international consensus statement
Transplantation
Cyclosporine microemulsion (Neoral) absorption profiling and sparse-sample predictors during the first 3 months after renal transplantation
Am J Transplant
The clinical benefits of cyclosporine C2-level monitoring: a systematic review
Transplantation
Opportunities to optimize tacrolimus therapy in solid organ transplantation: report of the European consensus conference
Ther Drug Monit
Efficacy and safety outcomes among de novo renal transplant recipients managed by C2 monitoring of cyclosporine a microemulsion: results of a 12-month, randomized, multicenter study
Transplantation
Results of a three-year prospective study of C2 monitoring in long-term renal transplant recipients receiving cyclosporine microemulsion
Transplantation
The temporal profile of calcineurin inhibition by cyclosporine in vivo
Transplantation
Inhibition of stimulated interleukin-2 production in whole blood: a practical measure of the cyclosporine effect
Clin Chem
Delayed cytokine mRNA expression kinetics after T-lymphocyte co-stimulation: a quantitative measure of the efficacy of cyclosporine A-based immunosuppression
Clin Chem
Sensitivity of whole-blood T lymphocytes in individual patients to tacrolimus (FK 506): impact of interleukin-2 mRNA expression as surrogate measure of immunosuppressive effect
Clin Chem
Calcineurin phosphatase activity in T lymphocytes is inhibited by FK 506 and cyclosporin A
Proc Natl Acad Sci U S A
FK-506- and CsA-sensitive activation of the interleukin-2 promoter by calcineurin
Nature
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2018, Developmental and Comparative ImmunologyCitation Excerpt :The absolute dependence on Ca2+/CaN signaling gives NFAT a remarkable ability to respond to [Ca2+]c increases and to sense Ca2+ oscillations in cells. Therefore, CaN/NFAT could critically control the gene expression implicated in cell differentiation and development (Sommerer et al., 2012; Müller and Rao, 2010). The NFAT complex also comprises another two DNA-binding proteins (heterodimer).
Cross talk among PMCA, calcineurin and NFAT transcription factors in control of calmodulin gene expression in differentiating PC12 cells
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