Urinary Time- or Dose-dependent Metabolic Biomarkers of Aristolochic Acid-induced Nephrotoxicity in Rats

The metabolic mechanisms underlying aristolochic acid (AA)-induced nephrotoxicity are inconclusive. A Gas Chromatography-Mass Spectrometer (GC-MS)-based metabolomic study was performed to analyze urinary metabolites in AA-treated rats at different dosages (10, 20, and 40 mg/kg) and time points (2, 4, and 6 days). Serum blood urea nitrogen (BUN), creatinine, and kidney injury were significantly changed only on the 6th day in 40 mg/kg AA group, whereas metabolic alternation appeared even on the 2nd day in 10 mg/kg AA group. A total of 84 differential metabolites were identified in 40 mg/kg AA groups time-dependently and 81 in 10, 20, and 40 mg/kg AA groups dose-dependently (6 days) compared with control group. Eight metabolites were selected as potential metabolic biomarkers including methylsuccinic acid, nicotinamide, 3-hydroxyphenylacetic acid, citric acid, creatinine, uric acid, glycolic acid, and gluconic acid. Four of them were dose-dependently altered including methylsuccinic acid, citric acid, creatinine, and 3-hydroxyphenylacetic acid, which were defined as "early metabolic biomarker." The alteration of nicotinamide, uric acid, and gluconic acid was time- and dose-dependent, whereas the change of glycolic acid was time- or dose-independent. The latter 4 metabolites were defined as "late metabolic biomarker" because of the obvious reduction on the 6th day in 40 mg/kg AA group. In summary, the urinary metabolic alterations were more sensitive than conventional biomarkers of renal injury. The identified metabolites suggested pathways of energy metabolism, gut microbiota, and purine metabolism were associated with AA-induced nephrotoxicity time- or dose-dependently. Further investigation was warranted to determine the roles of the 8 potential metabolic biomarkers in AA-induced nephrotoxicity.