# Caki-1 Spheroids as a Renal Model for Studying Free Fatty Acid-Induced Lipotoxicity

## TL;DR

This paper introduces a 3D cell model using Caki-1 spheroids to study how free fatty acids cause kidney cell damage, offering a new tool for understanding lipotoxicity in renal diseases.

## Contribution

The study introduces a novel 3D Caki-1 spheroid model for investigating free fatty acid-induced lipotoxicity in renal cells.

## Key findings

- Caki-1 spheroids formed stable 3D structures suitable for long-term culture.
- Palmitate exposure caused spheroid disintegration and cell death, which was mitigated by unsaturated fatty acids and acyl-CoA synthetase inhibition.
- Perilipin 2 knockdown worsened palmitate-induced cell death, highlighting its protective role.

## Abstract

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.