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

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.

Keywords: ER stress; autophagy; lipid droplet; lipotoxicity; tubular epithelial cells.

Figure 1

Formation and characterization of Caki-1 spheroids in a 3D culture model. (a) Representative images of Caki-1 spheroids captured on days 4, 6, 8, 10, and 12 after seeding. (b–d) Quantitative analysis of spheroid area (b), circularity (c), and roundness (d) from day 4 to day 12 using ImageJ software. Scale bar: 50 µm. Data represent mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. 30 spheroids were analyzed for three independent experiments.

Figure 2

Dose- and time-dependent effects of palmitate on Caki-1 spheroid morphology and cell viability. (a) Representative bright-field and live/dead staining images of Caki-1 spheroids following exposure to control (CON), 0.5% bovine serum albumin (BSA) only, or BSA-conjugated palmitate (PA) at concentrations of 100 µM, 200 µM, and 300 µM for 2 days. Live cells were stained in green with calcein AM and dead cells were stained in red with ethidium homodimer I. (b) Bright-field images showing time-dependent spheroid disintegration when 8-day-old (D0) spheroids were stimulated with high concentration (300 µM) of PA for 2 (D2) and 4 (D4) days. Scale bar: 50 µm. (c,d) Quantification of dead (c) and dissociated (d) cell number in spheroids treated with PA100–300 or OA300 for 2 days. (e) Quantification of the number of dissociated cells from the spheroids following fatty acid treatment for 2 and 4 days. Data represent mean ± SEM. ** p < 0.01 and **** p < 0.0001. 30 spheroids per group were analyzed for three independent experiments.

Figure 3

Palmitate-induced apoptosis in Caki-1 spheroids. (a) Western blot analysis of full-length PARP (PARP-pro, 116 kDa) and cleaved PARP (86 kDa) in Caki-1 spheroids treated with varying concentrations of palmitate [100 µM (PA100), 200 µM (PA200), and 300 µM (PA300)] or oleate [300 µM (OA300)] for 2 days. Data represent mean ± SEM. n = 3; ** p < 0.01. (b) Immunostaining for active caspase-3 (green) in Caki-1 spheroids following exposure to 300 µM of palmitate or oleate. Blue DAPI stains nuclei. Scale bar: 50 µm.

Figure 4

Inhibition of ACSL by triacsin C reduced palmitate-induced cytotoxicity in Caki-1 spheroids. (a) Representative live (green) and dead (red) staining images of Caki-1 spheroids treated with triacsin C (4 µM) for 1 h, followed by exposure to palmitate (300 µM) for 48 h. Palmitate (PA)-induced cell death was greatly attenuated in the presence of triacsin C (PA-TC). Scale bar: 50 µm. (b–f) Western blot analysis of PARP, LC3B, p62, and CHOP proteins in Caki-1 spheroids under the same treatment conditions. Palmitate-induced PARP cleavage (c) and upregulation of autophagy and ER stress markers LC3B-II (d), p62 (e), and CHOP (f) were significantly ameliorated by triacsin C. Data represent mean ± SEM. n = 3–4; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Figure 5

Oleate co-incubation protected against palmitate-induced cytotoxicity in Caki-1 spheroids. (a) Representative live (green) and dead (red) staining images of Caki-1 spheroids treated with BSA, palmitate (PA, 300 µM), or palmitate (300 µM) plus oleate (150 µM) (PA/OA) for 48 h. Oleate co-incubation substantially reduced the number of dead cells. Scale bar: 50 µm. (b–f) Western blot analysis of cleaved PARP, LC3B, p62, and CHOP in Caki-1 spheroids under the same treatment conditions. Oleate co-incubation greatly suppressed palmitate-induced PARP cleavage (c) and upregulation of autophagy and ER stress markers LC3B-II (d), p62 (e), and CHOP (f). Data represent mean ± SEM. n = 3–4; * p < 0.05, *** p < 0.001, and **** p < 0.0001.

Figure 6

Fatty acid treatment induced PLIN2 expression in Caki-1 spheroids. Western blot analysis showing a significant upregulation of PLIN2 expression in spheroids treated with high doses of palmitate and oleate compared to low-dose and control conditions. Data represent mean ± SEM. n = 3–4; ** p < 0.01 and *** p < 0.001.

Figure 7

PLIN2 knockdown enhanced PARP cleavage in BSA and palmitate-stimulated Caki-1 cells. (a) Representative Western blot images of PLIN2, PARP, CHOP, and p62 proteins in Caki-1 cells treated with BSA or palmitate (PA, 300 µM) for 24 h after transfection with negative siRNA (siNC) or siRNA targeting PLIN2/ADRP (siPLIN2). (b–e) Quantitative analysis of PLIN2 (b), cleaved PARP (c), CHOP (d), and p62 (e) proteins in BSA or PA-treated cells. Data represent mean ± SEM. n = 3–4; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.