Objectives: The host-microbiota co-metabolite trimethylamine N-oxide (TMAO) is linked to increased thrombotic and cardiovascular risks. Here we, sought to i) characterize which host variables contribute to fasting serum TMAO levels in real-life settings ii) identify potential actionable therapeutic means related to circulating TMAO. Design: We applied "explainable" machine learning, univariate-, multivariate- and mediation analyses of fasting plasma TMAO concentration and a multitude of bioclinical phenotypes in 1,741 adult Europeans of the MetaCardis study. We expanded and validated our epidemiological findings in mechanistic studies in human renal fibroblasts and a murine model of kidney fibrosis following TMAO exposure. Results: Next to age, kidney function was the primary variable predicting circulating TMAO in MetaCardis, with microbiota composition and diet playing minor, albeit significant roles. Mediation analysis revealed a causal relationship between TMAO and kidney function decline that strengthened at more severe stages of cardiometabolic disease. We corroborated our findings in preclinical models where TMAO exposure augmented conversion of human renal fibroblasts into myofibroblasts and increased kidney scarring in vivo. Mechanistically, TMAO aggravated kidney fibrosis due to ERK1/2 hyperactivation synergistically with TGF-β1 signaling. Consistent with our findings, patients receiving next-generation glucose-lowering drugs with reno-protective properties, had significantly lower circulating TMAO when compared to propensity-score matched control individuals. Conclusion: After age, kidney function is the major determinant of fasting circulating TMAO in adults. Our findings of lower TMAO levels in individuals medicated with reno-protective anti-diabetic drugs suggests a clinically actionable intervention for decreasing TMAO-associated excess cardiovascular risk that merits urgent investigation in human trials.

KC is a consultant for Danone Research, Ysopia and CONFO therapeutics for work not associated with this study. KC held a collaborative research contract with Danone Research in the context of MetaCardis project. FB is a shareholder of Implexion pharma AB. MB received lecture and/or consultancy fees from AstraZeneca, Boehringer-Ingelheim, Lilly, Novo Nordisk, Novartis and Sanofi.

This work was supported by European Union's Seventh Framework Program for research, technological development and demonstration under grant agreement HEALTH-F4-2012-305312 (METACARDIS). Assistance Publique-Hôpitaux de Paris (AP-HP) is the promoter of the clinical investigation (MetaCardis). Partial funding supports to K.C. and J.A.-W were also obtained from Leducq Foundation (TransAtlantic grant), SFN (Société Française de Nutrition), F-CRIN-FORCE network for support, INSERM via ITMO and JPI-Microdiet study and Novo Nordisk foundation (Jacobaus prize). S.K.F. received support from Deutsche Forschungsgesellschaft SFB1365 ("RENOPROTECTION") and SFB1470: "HFpEF". P.A. is an ISSF Fellow supported by the Wellcome Trust and Imperial College London (Grant No. ISSF 204834/Z/16/Z). K.Ch. is an ISSF Fellow supported by the Wellcome Trust and Imperial College London (Grant No. ISSF 204834/Z/16/Z). M.-E.D. is supported by the NIHR Imperial Biomedical Research Centre, GutsUK, Diabetes UK and by grants from the French National Research Agency (ANR-10-LABX-46, European Genomics Institute for Diabetes), from the National Center for Diabetes Precision Medicine - PreciDIAB, which is jointly supported by the French National Agency for Research (ANR-18-IBHU-0001), by the European Union (FEDER), by the Hauts-de-France Regional Council (Agreement 20001891/NP0025517) and by the European Metropolis of Lille (MEL, Agreement 2019_ESR_11) and by Isite ULNE (R-002-20-TALENT-DUMAS), also jointly funded by ANR (ANR16-IDEX-0004-ULNE), the Hauts-de-France Regional Council (20002845) and by the European Metropolis of Lille (MEL). This research was conducted within the context of the CNRS-Imperial International Research Project METABO-LIC. The Novo Nordisk Foundation Center for Basic Metabolic Research is an independent research institution at the University of Copenhagen partially funded by an unrestricted donation from the Novo Nordisk Foundation.

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Ethical approval was obtained from the Ethics Committee CPP lle-de France, the Ethical Committees of the Capital Region of Denmark (H-3-2013-145) and Ethics Committee at the Medical Faculty at the University of Leipzig (047-13-28012013).

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Raw shotgun sequencing data that support the findings of this study have been deposited in the European Nucleotide Archive with accession codes PRJEB37249, PRJEB38742, PRJEB41311 and PRJEB46098. Serum NMR and urine NMR metabolome data have been uploaded to Metabolights with accession number MTBLS3429; serum GC-MS and isotopically quantified serum metabolites (UPLC-MS/MS) are available from MassIVE with accession numbers MSV000088042 and MSV000088043, respectively.