Induction of Nephron-Ductal Dual Lineages via Early Retinoic Acid Signaling Establishes a Platform for Fully...

Kidney organoids are powerful tools for renal disease modeling and nephrotoxicity screening, yet their limited structural complexity-particularly the underdevelopment of ureteric bud (UB) lineages-remains a major limitation. A novel differentiation protocol is developed that short-term activation of retinoic acid (RA) signaling during the early intermediate mesoderm (IM) stage, enabling co-induction of anterior and posterior IM lineages. This eliminates the need for UB co-culture and supports the formation of kidney organoids containing complete nephron segments. BMP7 treatment during the maturation phase mimics UB-derived signals, further enhancing tubular maturation. Single-cell transcriptomic analysis confirms that RA activation promotes dual IM induction at early stages, while BMP7 enhances organoid maturity. These dual IMs enable the generation of tubule and collecting duct organoids, which serve as segment-specific models for renal reabsorption, efflux, and nephron-targeted drug response assessment. Furthermore, the organoids are integrated into a networking cell culture system with enterocytes and hepatocytes, establishing a physiologically integrated model for systemic drug evaluation. This platform enables more accurate in vitro analysis of drug absorption, metabolism, and nephrotoxicity, including NSAID-induced injury. Overall, this approach offers a robust, scalable, and physiologically relevant platform for next-generation renal modeling and pharmacological research.