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Indoxyl sulfate is a uremic toxin and a ligand of the aryl-hydrocarbon receptor (AhR), a transcriptional regulator. Elevated serum indoxyl sulfate levels may contribute to progressive kidney disease and associated vascular disease. We asked whether indoxyl sulfate injures podocytes in vivo and in vitro. Mice exposed to indoxyl sulfate for 8 w exhibited prominent tubulointerstitial lesions with vascular damage. Indoxyl sulfate-exposed mice with microalbuminuria showed ischemic changes, while more severely affected mice showed increased mesangial matrix, segmental solidification, and mesangiolysis. In normal mouse kidneys, AhR was predominantly localized to the podocyte nuclei. In mice exposed to indoxyl sulfate for 2 h, isolated glomeruli manifested increased Cyp1a1 expression, indicating AhR activation. After 8 w of indoxyl sulfate, podocytes showed foot process effacement, cytoplasmic vacuoles, and a focal granular and wrinkled pattern of podocin and synaptopodin expression. Furthermore, vimentin and AhR expression in the glomerulus was increased in the indoxyl sulfate-exposed glomeruli compared to controls. Glomerular expression of characteristic podocyte mRNAs was decreased, including Actn4, Cd2ap, Myh9, Nphs1, Nphs2, Podxl, Synpo, and Wt1. In vitro, immortalized-mouse podocytes exhibited AhR nuclear translocation beginning 30 min after 1 mM indoxyl sulfate exposure, and there was increased phospho-Rac1/Cdc42 at 2 h. After exposure to indoxyl sulfate for 24 h, mouse podocytes exhibited a pro-inflammatory phenotype, perturbed actin cytoskeleton, decreased expression of podocyte-specific genes, and decreased cell viability. In immortalized human podocytes, indoxyl sulfate treatment caused cell injury, decreased mRNA expression of podocytespecific proteins, as well as integrins, collagens, cytoskeletal proteins, and bone morphogenetic proteins, and increased cytokine and chemokine expression. We propose that basal levels of AhR activity regulate podocyte function under normal conditions, and that increased activation of podocyte AhR by indoxyl sulfate contributes to progressive glomerular injury. Citation: Ichii O, Otsuka-Kanazawa S, Nakamura T, Ueno M, Kon Y, et al. (2014) Podocyte Injury Caused by Indoxyl Sulfate, a Uremic Toxin and Aryl-Hydrocarbon Receptor Ligand. PLoS ONE 9(9): e108448. doi:10.1371/journal.pone. 0108448 Editor: Maria Pia Rastaldi, Fondazione IRCCS Ospedale Maggiore Policlinico & Fondazione D’Amico per la Ricerca sulle Malattie Renali, Italy Received May 6, 2014; Accepted August 21, 2014; Published September 22, 2014 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. For microarray data, this Minimum Information About a Microarray Experiment-compliant dataset has been deposited in the NCBI Gene Expression Omnibus, GEO Series accession number GSE51834 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? token = ezanqyoclzunxcf&acc = GSE51834). Funding: This work was partially supported by grants from the Institutional Program for Young Researcher Overseas Visits from the Japan Society for the Promotion of Science, a Grant-in-Aid for Young Scientists (A) (No. 24688033, Dr. Ichii) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and by the Intramural Research Program, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH) (Dr. Kopp). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: ichi-o@vetmed.hokudai. ac.jp