Three-dimensional–cultured renal tubular model reveals FXR-HNF4α-OAT1/3 axis as a target for preventing nucleotide analog–induced kidney injury

Drug-induced kidney injury, often resulting from the intracellular accumulation of drugs in renal proximal tubule cells via uptake transporters such as organic anion transporters 1 and 3 (OAT1/3), remains a major obstacle in drug development. Conventional 2-dimensional cultures of human renal proximal tubule epithelial cells (RPTECs) hardly express OAT1/3, limiting their utility for toxicity assessment. In contrast, 3-dimensional (3D) cultures of RPTEC have been shown to markedly upregulate OAT1/3 expression, offering a more physiologically relevant in vitro model for evaluating the toxicity of anionic compounds. In this study, we investigated the mechanism underlying OAT1/3 upregulation in 3D-RPTEC and explored a strategy to mitigate transporter-mediated toxicity. We found that hepatocyte nuclear factor (HNF) 4α expression is also increased in 3D-RPTEC. Motif analysis and cleavage under targets and release using nuclease-quantitative polymerase chain reaction revealed that HNF4α directly binds to the promoters of SLC22A6 and SLC22A8, identifying it as a key transcriptional regulator of OAT1/3 expression. Activation of the farnesoid X receptor (FXR), which represses the binding of HNF4α to promoters through the upregulation of small heterodimer partner (SHP), decreased OAT1/3 expression. Treatment with FXR ligands reduced cellular uptake of OAT1/3 substrates (eg, tenofovir and adefovir) and decreased their cytotoxic effects in 3D-RPTEC. These findings elucidate a transcriptional mechanism by which HNF4α regulates OAT1/3 expression in 3D-RPTEC and demonstrate that FXR agonists can downregulate OAT1/3 expression via the HNF4α-SHP axis. The present study highlights the utility of 3D-RPTEC as a valuable platform for mechanistic studies of transporter regulation. SIGNIFICANCE STATEMENT: This study shows that farnesoid X receptor agonists suppress hepatocyte nuclear factor 4α-mediated OAT1/3 activity in 3-dimensional-cultured renal proximal tubular cells and reduce nucleotide analog-induced toxicity. These findings provide mechanistic insight into transporter regulation and suggest a potential strategy to prevent nephrotoxicity.