Urine metabolomics links dysregulation of the tryptophan-kynurenine pathway to inflammation and severity of COVID-19

Abstract

SARS-CoV-2 causes major disturbances in serum metabolite levels, associated with severity of the immune response. Despite the numerous advantages of urine for biomarker discovery, the potential association between urine metabolites and disease severity has not been investigated in coronavirus disease 2019 (COVID-19). In a proof-of-concept study, we performed quantitative urine metabolomics in patients hospitalized with COVID-19 and controls using LC-MS/MS. We assessed whether metabolites alterations were associated with COVID-19, disease severity, and inflammation. The study included 56 patients hospitalized with COVID-19 (26 non-critical and 30 critical disease); 16 healthy controls; and 3 controls with proximal tubule dysfunction unrelated to SARS-CoV-2. Metabolomic profiling revealed a major urinary increase of tryptophan metabolites kynurenine (P < 0.001), 3-hydroxykynurenine (P < 0.001) and 3-hydroxyanthranilate (P < 0.001) in SARS-CoV-2 infected patients. Urine levels of kynurenines were associated with disease severity and systemic inflammation (kynurenine, r 0.43, P = 0.001; 3-hydroxykynurenine, r 0.44, P < 0.001). Increased urinary levels of neutral amino acids and imino acid proline were also common in COVID-19, suggesting specific transport defects. Urine metabolomics identified major alterations in the tryptophan-kynurenine pathway, consistent with changes in host metabolism during SARS-CoV-2 infection. The association between increased urinary levels of kynurenines, inflammation and COVID-19 severity supports further evaluation of these easily available biomarkers.

Figure 1

Urine Metabolomics in patients with COVID-19. (A) Study design. Urine samples obtained from patients with COVID-19 (n = 56, including 26 non-critical, 30 critical patients) were assessed using LC–MS/MS and compared to those obtained from healthy controls (n = 16 healthcare workers) and patients with proximal tubule (PT) dysfunction (n = 3) caused by tenofovir-related nephrotoxicity, Hartnup disease and Dent disease, respectively. Created in part with BioRender.com. (B) Heatmap of relative urinary concentrations of amino acids and metabolites in controls and patients hospitalized with COVID-19, stratified for disease severity. The 95th percentile of log-transformed values for each metabolite in healthy controls was considered as the reference. Patients with acute kidney injury (AKI) are identified by a black square on top of the heatmap.

Figure 2

Dysregulation of the tryptophan-kynurenine pathway in the urine of patients with COVID-19. (A) Volcano plot comparing log-transformed levels of urine metabolites in COVID-19 patients versus healthy controls. Significantly altered (increased) tryptophan metabolites are highlighted in red and the ten top dysregulated metabolites are identified with numbers and text. Two-sided Mann–Whitney U test followed by Benjamini and Hochberg multiple comparison test with false discovery rate (FDR) < 0.01. (B) Metabolites of the kynurenine pathway. Chemical structures were obtained from ChemSpider (www.chemspider.com), 2021. TDO, tryptophan 2,3-dioxygenase; IDO, indole 2,3-dioxygenase; KMO, kynurenine 3-monooxygenase; KYNU, kynureninase. (C) Comparison of log-transformed urinary levels of kynurenines and kynurenine to tryptophan ratio among controls (grey), non-critical (blue) and critical (red) COVID-19 patients. Data are individual values and medians. Comparisons using a one-way ANOVA followed by Holm-Sidak correction for multiple comparison. (D) Systemic inflammation, assessed by the plasma level of C-reactive protein (CRP) at the time of sampling, correlates with urinary (ur.) concentration of kynurenine, 3-hydroxykynurenine and the kynurenine to tryptophan ratio (KTR) in COVID-19. Data are individual values (black dots), and linear regressions with 95% confidence intervals (red lines).

Figure 3

Prevalence and pattern of aminoaciduria in COVID-19. (A) Heatmap of aminoaciduria in COVID-19 patients. Presence (dark blue) of aminoaciduria was defined as a concentration above the 95th percentile of log-transformed values in healthy controls. Dark blue (vs. light blue) cells indicate presence (vs. absence) of aminoaciduria, for the 20 classical amino acids. Patients with acute kidney injury (AKI) are identified by a black square on top of the heatmap. Bas., basic; Ac., acidic. (B) Prevalence of aminoaciduria in COVID-19. Neutral, basic and acidic amino acids are indicated in green, blue and red, respectively. (C) Comparison of log-transformed urinary levels of the most prevalent amino acids among controls (grey), non-critical (blue) and critical (red) COVID-19 patients. Data are individual values and medians. Comparisons using a one-way ANOVA followed by Holm-Sidak correction for multiple comparison.