Non-steroidal anti-inflammatory drugs (NSAIDs) are often the first-line approach to the treatment of pain and inflammatory conditions. The NSAID indomethacin is commonly used to manage pain in osteoarthritis, rheumatoid arthritis, degenerative joint diseases, and gout [1]. Its mechanism of action is the inhibition of prostaglandin synthesis. This is achieved by suppressing the activity of cyclooxygenase, which is a crucial regulator of fever, pain, and inflammation [2]. Prostaglandin E2 (PGE2) is an important immunopathologic modulator with immune regulatory effects in chronic infections such as tuberculosis (TB) [3]. Host-directed treatment of TB, in which PGE2 is reduced after administration of indomethacin, may be an option for treating clinically complicated or resistant TB [4] in combination with anti-TB drugs. However, the clinical use of indomethacin has been limited by the high incidence and severity of adverse reactions to the drug [5]. The molecular mechanisms behind the undesirable effects of indomethacin have not been well studied.

Indomethacin causes systemic toxicity in the kidney, liver, and intestine [5,6]. In an in vivo rat experiment, indomethacin significantly affected several biochemical serum parameters [5]. Moreover, significantly increasing total bilirubin, alanine aminotransferase, and aspartate aminotransferase (AST) (indicative of liver damage) [7], as well as serum blood urea nitrogen (BUN) and serum creatinine (CREA) (indicating kidney damage and risk of acute kidney injury, tubulointerstitial nephritis, nephrotic syndrome, and chronic kidney disease) were reported [8,9]. Moreover, PG depletion in the kidneys reduces prostacyclin and PGE2, which are major vasodilators, resulting in severe renal vasoconstriction and, by extension, irreversible renal ischemia and acute tubular necrosis [10]. Indomethacin also induces oxidative stress in the kidney through the depletion of heme. The resulting reduction in the level of renal endothelial nitric oxide (NO) decreases renal perfusion, leading to glomerular and tubular impairment and renal damage [11]. A significant decrease in superoxide dismutase (SOD) and catalase, which are the primary cellular defenses against reactive oxygen species (ROS) [5], can cause an imbalance in cellular oxidant-antioxidant status [12] and damage cell membranes via lipid peroxidation [13]. The accumulation of superoxide by mitochondria increases the susceptibility of the kidney to oxidative stress-induced damage, leading to progressive renal impairment [14,15]. Recent studies have shown that increased ROS levels and lipid peroxidation trigger ferroptosis, which plays an important role in pathophysiological processes involving the kidney [16]. In addition, indomethacin stimulates the protein kinase Cζ -p38 MAPK-dynamin-related protein 1 pathway; this disrupts mitochondrial dynamics, resulting in apoptosis [17]. However, whether ferroptosis or apoptosis is involved in the adverse effects of indomethacin on non-target organs, such as the kidney and liver, remains unknown. Mechanistic insights into indomethacin toxicity may allow for improved formulations and therefore an expansion of the drug's clinical applications.

Multi-omics studies, such as those combining metabolomics and transcriptomics, as well as analyses of endogenous metabolites in serum and urine, may shed light on the toxic effects of indomethacin. In a previous study, we conducted a comprehensive genome-wide analysis of the functional pathways associated with nephrotoxic agents, to identify common and distinct molecular processes [18]. We found that repeated exposure to indomethacin (10 mg/kg) induced a large number of transcriptional alterations in the kidney and liver of rats. The changes included up-regulated expression of genes involved in the cell cycle, lipid metabolism and atherosclerosis, and the TNF signaling pathway, and down-regulated expression of genes involved in retinol metabolism, the citrate cycle (TCA cycle), and oxidative phosphorylation. However, the focus of the study was on the common and distinct molecular processes of some commonly used nephrotoxic drugs. In the present study, the molecular alterations in the kidney, liver, urine, and serum induced by indomethacin were further explored by analyzing the respective metabolic profile, and direct readouts of phenotypes. We also combined transcriptomics data for pathway-level multi-omics information, to better delineate the molecular mechanisms responsible for indomethacin toxicity.