Establishment of a Drug Toxicity Assessment Using Kidney Organoids

Background: Efforts to reduce animal testing under the 3R principles (replacement, reduction and refinement) have highlighted new approach methodologies. Here, we developed a kidney organoid system derived from adult rat kidney stem (KS) cells and evaluated its utility as a screening platform for nephrotoxicity. Furthermore, we applied this system to assess the nephrotoxic effects of puberulic acid (PA), a compound suspected to be associated with acute kidney injury (AKI) cases linked to Beni-koji Cholestehelp supplements recently reported in Japan. Methods: Nephrotoxicity was evaluated in KS cell-derived kidney organoids treated with known nephrotoxic agents, cisplatin and gentamicin for 72 hours. Assessment included histological analysis and qPCR analysis of kidney injury molecule-1 (Kim-1), a biomarker for AKI. Additionally, the nephrotoxicity of PA was investigated using both the kidney organoid model and an in vivo mouse model. C57BL/6N mice were administered PA intraperitoneally, and renal function and histopathological findings were evaluated after 4 days. Results: In kidney organoids, treatment with cisplatin and gentamicin induced significant apoptosis, demonstrated by an increase in cleaved caspase-3–positive cells. Transmission electron microscopy revealed loss of intercellular junctions and cytoplasmic vacuolization, characterized as acute tubular necrosis (ATN). qPCR analysis showed a significant upregulation of Kim-1 expression in organoids treated with cisplatin and gentamicin. Similarly, exposure to PA resulted in proximal tubular injury in kidney organoids, accompanied by increased Kim-1 expression and a higher number of cleaved caspase-3–positive apoptotic cells. In vivo, PA-treated mice demonstrated impaired renal function, evidenced by elevated serum creatinine levels and an increased urinary albumin-to-creatinine ratio. Histological analysis revealed tubular atrophy, cell shedding and cast formation without glomerular abnormalities, consistent with ATN, paralleling the findings observed in organoids. Conclusion: This study demonstrated the quantitative evaluation of renal tubular damage using kidney organoids and revealed the nephrotoxicity of PA in supplement-induced AKI. The KS cell-derived kidney organoid system offers a cost-effective, reproducible platform for nephrotoxicity screening as an alternative to animal models. Funding: Private Foundation Support