Targeted Metabolomics Reveals Hippurate as a Urinary Potential Marker for Diabetic Nephropathy

Background: Targeted urinary metabolic profiling of patients with type 2 diabetes mellitus (DM) and diabetic nephropathy (DN) along with healthy control was conducted with urine being a non-invasive biofluid and ideal for the biomarker studies related to kidney disorders. Objective: Other markers of risk for DN in combination with microalbuminuria are needed for optimal clinical management. The purpose of this study was to propose new urinary metabolic markers involved in the pathophysiology the DN. Method: Liquid chromatography coupled mass spectrometry (LC-MS) method was developed using diamond hydride column for 43 diverse polarities of metabolites. Multiple reaction monitoring (MRM) was performed using optimized ionization modes for the 43 studied metabolites. Diabetics (n=26), diabetic nephropathy (n=27) patients, and healthy controls (27) participated in the study and chemometric analysis was performed using Graphpad and non-commercial software "Multibase excel add in" to identify significant metabolites. Pathway analysis is performed using free software metaboanalyst. Results: Screening of metabolites with the Bonferroni multiple comparison test was performed, which suggested significantly higher urinary excretion of hippurate in the DN patients as compared to DM and the control group. Significant positive correlation was found between hippurate and albumin across all the three groups. Partial least square discriminant analysis (PLS-DA) was performed for DN and DM groups and showed a good separation between DM patients and DN patients. Further, the pathway analysis suggested the involvement of arginine and proline metabolism in the pathogenesis of DN. Conclusion: This study suggests potential use of new LC-MS method for targeted analysis of metabolites covering different pathways and proposes hippurate potential urinary marker metabolites for discrimination of DM and DN groups.