Nakanoh, H.; Tsuji, K.; Uchida, N.; Fukushima, K.; Haraguchi, S.; Kitamura, S.; Wada, J.

Considerable efforts have been made to reduce animal experimentation in accordance with the 3R principles; replacement, reduction, and refinement; thus New Approach Methodologies have emerged as an urgent necessity. In this context, we developed a kidney organoid system derived from adult rat kidney stem (KS) cells and evaluated its potential as a screening platform for nephrotoxicity. Nephrotoxicity was assessed in KS cell-derived organoids treated with known nephrotoxic agents, including cisplatin and gentamicin. Additionally, we investigated the nephrotoxicity of puberulic acid, a compound associated with acute kidney injury linked to the Beni-koji CholesteHelp supplement recently reported in Japan, using both kidney organoids and wild-type mice. The organoid model successfully recapitulated morphological injury induced by known nephrotoxins and enabled quantitative assessment via Kidney Injury Molecule-1 (Kim-1) mRNA expression. In organoids, puberulic acid induced apoptosis, demonstrated by increased cleaved caspase-3-positive cells, similar to cisplatin-treated samples. Transmission electron microscopy revealed structural alterations such as loss of intercellular tight junctions and vacuolization, consistent with acute tubular necrosis (ATN). Quantitative real-time PCR analysis confirmed significant Kim-1 upregulation following nephrotoxic exposure. In vivo, puberulic acid-treated mice exhibited impaired renal function and histological findings of ATN, closely resembling the organoid results. This KS cell-derived kidney organoid system offers a reproducible and rapid screening tool for nephrotoxicity with simple protocol. The ability to assess both morphological and quantitative changes following drug exposure highlights the utility of this organoid platform for toxicological evaluation and supports its potential as an alternative to animal models.