Urinary metabolomic profiling from spontaneous tolerant kidney transplanted recipients shows enrichment in tryptophan-derived metabolites

Abstract

Background: Operational tolerance is the holy grail in solid organ transplantation. Previous reports showed that the urinary compartment of operationally tolerant recipients harbor a specific and unique profile. We hypothesized that spontaneous tolerant kidney transplanted recipients (KTR) would have a specific urinary metabolomic profile associated to operational tolerance.

Methods: We performed metabolomic profiling on urine samples from healthy volunteers, stable KTR under standard and minimal immunosuppression and spontaneous tolerant KTR using liquid chromatography in tandem with mass spectrometry. Supervised and unsupervised multivariate computational analyses were used to highlight urinary metabolomic profile and metabolite identification thanks to workflow4metabolomic platform.

Findings: The urinary metabolome was composed of approximately 2700 metabolites. Raw unsupervised clustering allowed us to separate healthy volunteers and tolerant KTR from others. We confirmed by two methods a specific urinary metabolomic signature in tolerant KTR mainly driven by kynurenic acid independent of immunosuppressive drugs, serum creatinine and gender.

Interpretation: Kynurenic acid and tryptamine enrichment allowed the identification of putative pathways and metabolites associated with operational tolerance like IDO, GRP35 and AhR and indole alkaloids.

Funding: This study was supported by the ANR, IRSRPL and CHU de Nantes.

Keywords: Kidney transplantation; Kynurenic acid; Metabolomic; Operational tolerance; Tryptophan; Urine.

Figure 1

Schematic representation of the workflow used to identify the metabolomic signature of operational tolerance. This figure was created by BioRender.

Figure 2

Richness and structure of the urinary metabolome for each group of KTR and HV with RP UHPLC-MS method. (a) Chromatogram showing showing 2161 ions and 2681 ions with a major proportion of highly polar and polar metabolites in ESI+ and ESI- modes respectively according acetonitrile gradient and retention time (RT). (b) Structure of urinary metabolome in ESI+ assessed by principal component analysis (PCA) with the first three components for recipient groups (TOL, MIS, STA, scABMR) and healthy volunteers revealing two clusters: one grouping TOL and HV and another grouping MIS, STA and scABMR. (c) Structure of the urinary metabolome in ESI- assessed by principal component analysis (PCA) with the first three components for recipient groups (TOL, MIS, STA, scABMR) and healthy volunteers revealing an isolated cluster of HV in the three PCs and KTR clustering in roughly parallel planes from TOL/MIS/STA/scABMR.

Figure 3

Specific metabolomic signature in urine of TOL detected thanks to RP UHPLC-MS method. (a) represents the supervised clustered heatmap according to KTR (TOL, nonTOL) and HV of the twelve ions composing the specific urinary signature of TOL patients where ten are upregulated in TOL (red cluster) and two are downregulated in TOL (black to blue cluster). Among the twelve ions, four were identified as being adducts of kynurenic acid (highlighted in red) (b) as shown in boxplots (c) which allow a good discrimination of TOL compared to nonTOL patients according to the ROCC. * indicates an FDR-adjusted p-value < 0.1; ** indicates an FDR-adjusted p-value < 0.01 and ***indicates an FDR-adjusted p-value < 0.001

Figure 4

Tryptophan-derived metabolites detected in the urine samples of our cohort (TOL, nonTOL and HV) and their associated metabolic pathways detected thanks to RP UHPLC-MS method. Kynurenine, kynurenic acids and tryptamine were upregulated in TOL compared to nonTOL and HV as shown in boxplots. Solid lines represent detected and identified metabolites; dashed lines represent nonidentified metabolites; grey shading indicates no change in TOL; blue-shading indicates downregulation; red shading indicates upregulation; * indicates an FDR-adjusted p-value < 0.1; ** indicates an FDR-adjusted p-value < 0.01 and *** indicates an FDR-adjusted p-value < 0.001