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The poorly soluble metabolite 7\u2010hydroxymethotrexate contributes to renal injury, potentially through intratubular precipitation; however, its formation differs across species, complicating the evaluation of human\u2010relevant toxicity in conventional animal models. In this study, methotrexate\u2010induced renal toxicity was investigated using chimeric mice with humanized livers (humanized\u2010liver mice). The pharmacokinetics of methotrexate and its major metabolite, 7\u2010hydroxymethotrexate, were characterized in humanized\u2010liver mice and compared with those in nontransplanted mice. Following the administration of a single oral dose, plasma concentrations and area under the curves of methotrexate and 7\u2010hydroxymethotrexate were higher in humanized\u2010liver mice than in control mice, and the urinary metabolite\u2010to\u2010parent ratio in humanized\u2010liver mice was comparable to that reported in humans. Repeated methotrexate administration significantly increased blood urea nitrogen and creatinine levels, indicating impaired renal function. Necrosis of tubular epithelial cells and eosinophilic debris within the tubules were observed in the kidneys of humanized\u2010liver mice, along with the accumulation of methotrexate and 7\u2010hydroxymethotrexate, but not in control mice. Hepatic cytosolic methotrexate 7\u2010hydroxylation activity in humanized\u2010liver mice showed kinetic properties and inhibitor sensitivity consistent with human aldehyde oxidase, supporting this enzyme's role in metabolite formation. Thus, humanized\u2010liver mice recapitulate key features of methotrexate\u2010induced renal toxicity associated with human\u2010relevant metabolism, providing a useful in\u00a0vivo platform to evaluate organ toxicity in the context of human pharmacokinetics.<\/jats:p>","DOI":"10.1002\/jat.70451","type":"journal-article","created":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T06:53:16Z","timestamp":1790664796000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Association of Methotrexate\u2010Induced Renal Toxicity in Humanized\u2010Liver Mice With Human AOX1\u2010Derived 7\u2010Hydroxymethotrexate"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-5020-3853","authenticated-orcid":false,"given":"Yuki","family":"Obata","sequence":"first","affiliation":[{"name":"Liver Engineering Laboratory, Department of Research for Humanized Model Central Institute for Experimental Medicine and Life Science  Kawasaki Kanagawa Japan"},{"name":"Department of Environmental Health Sciences Hoshi University  Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1359-8828","authenticated-orcid":false,"given":"Shotaro","family":"Uehara","sequence":"additional","affiliation":[{"name":"Liver Engineering Laboratory, Department of Research for Humanized Model Central Institute for Experimental Medicine and Life Science  Kawasaki Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuichiro","family":"Higuchi","sequence":"additional","affiliation":[{"name":"Liver Engineering Laboratory, Department of Research for Humanized Model Central Institute for Experimental Medicine and Life Science  Kawasaki Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-1936-0425","authenticated-orcid":false,"given":"Masahiko","family":"Yasuda","sequence":"additional","affiliation":[{"name":"Pathology Center Central Institute for Experimental Medicine and Life Science  Kawasaki Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takaya","family":"Homma","sequence":"additional","affiliation":[{"name":"Liver Engineering Laboratory, Department of Research for Humanized Model Central Institute for Experimental Medicine and Life Science  Kawasaki Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yukari","family":"Totsuka","sequence":"additional","affiliation":[{"name":"Department of Environmental Health Sciences Hoshi University  Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3706-2454","authenticated-orcid":false,"given":"Hiroshi","family":"Suemizu","sequence":"additional","affiliation":[{"name":"Liver Engineering Laboratory, Department of Research for Humanized Model Central Institute for Experimental Medicine and Life Science  Kawasaki Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,28]]},"reference":[{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.5455\/jice.20161208013809"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1124\/dmd.112.044545"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1124\/dmd.123.001257"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1111\/cts.13125"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1124\/dmd.119.086579"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/cam4.70516"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.1002\/1097\u20100142(197801)41:1<36::aid\u2010cncr2820410108>3.0.co;2\u2010i"},{"issue":"1","key":"e_1_2_9_9_1","first-page":"1","article-title":"A Specific HPLC Assay to Determine the Pharmacokinetics of Methotrexate in Patients","volume":"22","author":"Chen M. 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