Battle Dana, Qiu Xiangzhe, Alex Marilyn, Rivers London, Hamilton Jamie A G, Takayama Shuichi, Zhao Xueying
Department of Physiology, Morehouse School of Medicine, Atlanta, GA 30310, USA.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA 30332, USA.
Cells. 2025 Feb 27;14(5):349. doi: 10.3390/cells14050349.
Lipotoxicity, resulting from the buildup of excess lipids in non-adipose tissues, is increasingly recognized as a major contributor to the progression of kidney disease, highlighting the need for alternative models to assess its effects on renal cells. 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 4-12. 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.
脂毒性是由非脂肪组织中过量脂质蓄积所致，日益被认为是肾病进展的主要促成因素，这凸显了需要有替代模型来评估其对肾细胞的影响。本研究的主要目的是探讨人近端小管（PT）和肾细胞癌（RCC）代表性细胞系Caki-1作为研究游离脂肪酸诱导的PT脂毒性的三维模型系统的实用性。在超低附着板上生成并维持Caki-1球体，并对其随时间变化的形态变化进行表征。在最佳三维培养条件下，Caki-1细胞形成形态明确、紧密的大球体，形态均匀、圆度良好，直径在第4至12天增大。长期暴露于饱和棕榈酸会导致球体呈剂量和时间依赖性解体及细胞死亡，包括球体形态分散和平坦化，外周层死细胞增多，球体核心减小。此外，经棕榈酸处理的球体中裂解的聚（ADP-核糖）聚合酶（PARP）和活性半胱天冬酶-3显著增加。长链酰基辅酶A合成酶抑制剂三辛素C可减轻棕榈酸诱导的PARP裂解以及内质网（ER）应激和自噬功能障碍。此外，与不饱和油酸共同孵育可防止棕榈酸诱导的Caki-1三维培养中的球体解体和凋亡细胞死亡。虽然脂肪酸过载上调了Caki-1细胞中的脂滴蛋白围脂滴蛋白2，但通过小干扰RNA敲低围脂滴蛋白2会导致棕榈酸诱导的细胞死亡加剧。总之，这些结果表明三维Caki-1球体模型是一种简单且可重复的体外系统，用于研究肾脂毒性和脂质代谢，能在分子、细胞和多细胞水平提供有用的读数。
