World-wide exposure to aristolochic acid (AA) via contaminated wheat grain and Chinese herbal medicines is substantial (~100 million), with a significant number of exposed individuals (~5%) succumbing to AA nephropathy (AAN). The mechanism of AA-induced nephrotoxicity, and the molecular basis for susceptibility to AAN remain unknown. AA is metabolized via CYP primarily via O-demethylation to the corresponding hydroxyl metabolite, AA1a, and by nitroreductases to a mutagenic N-hydroxy intermediate. Metabolic pathways responsible for the bioactivation of AA to nephrotoxic species have not been identified. We hypothesize that hepatic CYP(s) open the methylenedioxy moiety of AA yielding a catechol metabolite that can be scavenged by glutathione and subsequently targeted to renal proximal tubule epithelial cells (the physiological target of AA). We will therefore employ the fabricated liver-to-kidney on-a-chip design to test the hypothesis that liver-derived metabolites of AA are responsible for AA-mediated nephrotoxicity. Preliminary data will provide the basis for two potential NIEHS grant applications; a R21 to further refine the technology to a high throughput capability for the testing of environmental chemicals and for expanding the potential organ/tissue pairs (or >2), and one (R21) to utilize the model to examine the toxicity of AA. The R21 will provide data for a subsequent RO1 application which will elaborate on the molecular mechanism(s) by which nephrotoxic metabolites of AA induce toxicity, a prerequisite for identifying susceptibility factors that can be applied in paradigms of prevention, diagnosis, and therapy.