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The main aim of this study was to investigate the usefulness of Caki-1, a human proximal tubule (PT) and renal cell carcinoma (RCC) representative cell line, as a 3D model system for studying free fatty acid-induced PT lipotoxicity. Caki-1 spheroids were generated and maintained on ultra-low attachment plates and characterized regarding time-dependent morphology changes. In optimal 3D culture conditions, Caki-1 cells formed well-defined large compact spheroids with uniform morphology, good circularity, and increased diameter from days 4\u201312. Chronic exposure to saturated palmitate resulted in dose- and time-dependent spheroid disintegration and cell death, including dispersed and flattened spheroid morphology, with increased dead cells in the peripheral layers and decreased spheroid core. Moreover, palmitate-treated spheroids showed a significant increase in cleaved poly(ADP-ribose) polymerase (PARP) and active caspase-3. Palmitate-induced PARP cleavage, as well as endoplasmic reticulum (ER) stress and autophagy dysfunction, were blunted by triacsin C, an inhibitor of long-chain acyl-CoA synthetases. In addition, co-incubation with unsaturated oleate prevented palmitate-induced spheroid disintegration and apoptotic cell death in Caki-1 3D culture. While fatty acid overload upregulated lipid droplet protein perilipin 2 in Caki-1 cells, knockdown of perilipin 2 by siRNAs resulted in an exacerbation of palmitate-induced cell death. Together, these results indicate that the 3D Caki-1 spheroid model is a simple and reproducible in vitro system for studying renal lipotoxicity and lipid metabolism that gives useful readouts at the molecular, cellular, and multicellular levels.<\/jats:p>","DOI":"10.3390\/cells14050349","type":"journal-article","created":{"date-parts":[[2025,2,28]],"date-time":"2025-02-28T04:28:18Z","timestamp":1740716898000},"page":"349","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Caki-1 Spheroids as a Renal Model for Studying Free Fatty Acid-Induced Lipotoxicity"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0001-6311-7850","authenticated-orcid":false,"given":"Dana","family":"Battle","sequence":"first","affiliation":[{"name":"Department of Physiology, Morehouse School of Medicine, Atlanta, GA 30310, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiangzhe","family":"Qiu","sequence":"additional","affiliation":[{"name":"Department of Physiology, Morehouse School of Medicine, Atlanta, GA 30310, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0005-9669-0791","authenticated-orcid":false,"given":"Marilyn","family":"Alex","sequence":"additional","affiliation":[{"name":"Department of Physiology, Morehouse School of Medicine, Atlanta, GA 30310, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"London","family":"Rivers","sequence":"additional","affiliation":[{"name":"Department of Physiology, Morehouse School of Medicine, Atlanta, GA 30310, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3071-6029","authenticated-orcid":false,"given":"Jamie A. 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