Development of a Liver-Kidney Dual Organ Microphysiological System for Predicting Organ-Organ Interactions in the Toxicity of Xenobiotics: Aristolochic Acid Nephrotoxicity as a proof of Principle

Development of a Liver-Kidney Dual Organ Microphysiological System for Predicting Organ-Organ Interactions in the Toxicity of Xenobiotics: Aristolochic Acid Nephrotoxicity as a proof of Principle Shih-Yu Chang Chair of the Supervisory Committee: Professor David L. Eaton Department of Environmental and Occupational Health Sciences The liver is the principal organ responsible for drug and xenobiotics metabolism, including inactivation or bioactivation. To improve the predictability of drug safety and efficacy in clinical development, and to facilitate the evaluation of the potential human health effects from exposure to environmental contaminants, there is a critical need to accurately model human organ systems such as the liver in vitro. Currently, there are numerous limitations of in vitro cell culture models. We developed a microphysiological system (MPS) based on a new commercial microfluidic platform (Nortis Inc., Woodinville, WA) that can utilize primary liver cells from mammals (e.g., rat and human). Compared to conventional monolayer cell culture which typically survives for 5-7 days or less, primary rat or human hepatocytes in MPS exhibited: higher viability, improved hepatic functions, such as albumin production, expression of hepatocyte marker HNF4α and canaliculi structure, up to 14 days and longer. Additionally, induction of cytochrome P450 (CYP) 1A and 3A4 in human hepatocytes was observed in MPS. These results indicate that hepatocytes cultured in MPS provide a promising approach for evaluating chemical toxicity in vitro. Additionally, to test the hypothesis that hepatic clearance of a nephrotoxic chemical might have significant importance in determining ultimate kidney toxicity, we utilized aristolochic acid-I (AA-I), a well-known nephrotoxin and carcinogen, that undergoes extensive hepatic metabolism. We also developed an integrated MPS model with an interconnected liver-on-a-chip populated with hepatocytes , and a kidney-on-a-chip platform using proximal tubule epithelial cells (PTECs). Our results of this proof of concept study demonstrated that hepatocyte-dependent metabolism of AA-I prior to PTEC exposure substantially increased cytotoxicity to PTECs, formation of aristolactam-I (AL-I) DNA adducts in PTECs, and release of KIM-1 and other organ injury biomarkers, indicating that hepatic metabolism apparently contributes more to bioactivation of AA-I than to its detoxification. Additional mechanistic studies provided mechanistic insights into the important role of hepatic biotransformation for the kidney-specific toxicity of AA-I, potentially involved with activation to the AA-lactam intermediate via NADPH:quinone oxidoreductase (NQO1), sulfate conjugation via hepatic sulfotransferases (SULTs), and hepatic export and renal uptake via organic anion transporters (OATs) uptake. This in vitro/ex vivo integrated organs-on-chips culture can be used to identify toxicologically relevant organ-organ interactions that may occur in vivo, providing a novel approach for investigating the mechanisms that underlay toxicologically important organ-organ interactions.