Targeted Metabolomics Analysis Suggests That Tacrolimus Alters Protection against Oxidative Stress

Abstract

Tacrolimus (FK506) is an immunosuppressant that is experiencing a continuous rise in usage worldwide. The related side effects are known to be globally dose-dependent. Despite numerous studies on FK506, the mechanisms underlying FK506 toxicity are still not well understood. It is therefore essential to explore the toxicity mediated by FK506. To accomplish this, we conducted a targeted metabolomic analysis using LC-MS on the plasma samples of patients undergoing FK506 treatment. The aim was to identify any associated altered metabolic pathway. Another anti-calcineurin immunosuppressive therapy, ciclosporin (CSA), was also studied. Increased plasma concentrations of pipecolic acid (PA) and sarcosine, along with a decrease in the glycine/sarcosine ratio and a tendency of increased plasma lysine was observed in patients under FK506 compared to control samples. Patients under CSA do not show an increase in plasma PA compared to the control samples, which does not support a metabolic link between the calcineurin and PA. The metabolomics changes observed in patients under FK506 highlight a possible link between FK506 and the action of an enzyme involved in both PA and sarcosine catabolism and oxidative pathway, the Peroxisomal sarcosine oxidase (PIPOX). Moreover, PA could be investigated as a potential biomarker of early nephrotoxicity in the follow-up of patients under FK506.

Keywords: immunosuppressive; metabolomics; oxidative stress; pipecolic acid; tacrolimus.

Figure 1

Unbiased analysis of plasma amino acid of patients with and without FK506. (A) Plot with Pearson correlation coefficients for each amino acid along the two major axis of the PCA; (B) Score plot of the groups along the two major axes of the PCA with 95% confidence ellipses; (C) Variable importance plot of the Random Forest analysis. The variables are ordered top-to-bottom as most-to-least important for classifying between the two groups; (D) Enrichment metabolite sets from separating more metabolites.

Figure 2

Plasma pipecolic acid increase in case of FK506 treatment independently of calcineurin pathway. (A) Plasma pipecolic concentrations according to different groups (FK506 vs Control). (B) Plasma pipecolic concentrations according to different groups (CSA vs Control). (C) Plasma pipecolic concentrations according to different groups (FK506 vs CSA) Control (n = 7), FK506 (n = 19) and CSA (n = 9). Data are expressed as median ± IQR. p-values are obtained using the Mann–Whitney test.

Figure 3

FK506 seems to alter the metabolic pathways involving PIPOX. (A) Plasma Lysine concentrations according to the different groups: Control (n = 7) and FK506 (n = 19). (B) Plasma Sarcosine concentrations according to the different groups: Control (n = 7) and FK506 (n = 19). (C) Plasma Serine, concentrations according to the different groups: Control (n = 7) and FK506 (n = 19). (D) Plasma Glycine/Sarcosine concentrations ratio according to the different groups: Control (n = 7) and FK506 (n = 19). Data are expressed as median ± IQR. p-values are obtained using the Mann–Whitney test.

Figure 4

Summary of the metabolic changes induced by Tacrolimus. The red arrows indicate significant plasma variations, the blue arrows indicate non-significant plasma variation trends. PIPOX: pipecolate oxidase. SARDH: sarcosine oxidase. P5CR: pyrroline-5-carboxylate reductase. SHMT1: serine hydroxymethyltransferase. GNMT: glycine N-methyltransferase.