Ezetimibe Enhances Lipid Droplet and Mitochondria Contact Formation, Improving Fatty Acid Transfer and Reducing Lipotoxicity in Alport Syndrome Podocytes

Abstract

Mitochondrial dysfunction is a critical factor in the pathogenesis of Alport syndrome (AS), contributing to podocyte injury and disease progression. Ezetimibe, a lipid-lowering drug, is known to inhibit cholesterol and fatty acid uptake and to reduce triglyceride content in the kidney cortex of mice with AS. However, its effects on lipid droplet (LD) utilization by mitochondria have not been explored. Transmission electron microscopy (TEM) and mitochondrial functional assays (ATP production, mitochondrial membrane potential, and citrate synthase activity) were used to investigate the impact of ezetimibe on LD-mitochondria contact formation and mitochondrial function in Col4a3KO (AS) and wildtype (WT) podocytes. TEM analysis revealed significant mitochondrial abnormalities in AS podocytes, including swollen mitochondria and reduced cristae density, while mitochondrial function assays showed decreased ATP production and lowered mitochondrial membrane potential. AS podocytes also demonstrated a higher content of LD but with reduced LD-mitochondria contact sites. Ezetimibe treatment significantly increased the number of LD-mitochondria contact sites, enhanced fatty acid transfer efficiency, and reduced intracellular lipid accumulation. These changes were associated with a marked reduction in the markers of lipotoxicity, such as apoptosis and oxidative stress. Mitochondrial function was significantly improved, evidenced by increased basal respiration, ATP production, maximal respiration capacity, and the restoration of mitochondrial membrane potential. Additionally, mitochondrial swelling was significantly reduced in ezetimibe-treated AS podocytes. Our findings reveal a novel role for ezetimibe in enhancing LD-mitochondria contact formation, leading to more efficient fatty acid transfer, reduced lipotoxicity, and improved mitochondrial function in AS podocytes. These results suggest that ezetimibe could be a promising therapeutic agent for treating mitochondrial dysfunction and lipid metabolism abnormalities in AS.

Keywords: Alport syndrome podocytes; fatty acid; lipid droplet; lipotoxicity.

Figure 1

Increased TG lipolysis in immortalized AS podocytes is associated with mitochondrial dysfunction and podocyte injury due to inefficient FA transfer. (A) Lipolysis assay demonstrating significantly increased lipolysis in immortalized AS podocytes when compared to immortalized WT podocytes (n = 3, * p < 0.05, t-test). (B) Apoptosis increases in immortalized AS podocytes when compared to immortalized WT podocytes (n = 3–4, *** p < 0.001, t-test). (C,D) TEM image analysis demonstrates lower mitochondria and LD interaction in immortalized AS podocytes compared to immortalized WT podocytes; red arrow indicates LD contact with mitochondria, and blue arrow indicates distance between LD and mitochondria (C), quantified (D) (* p < 0.05, t-test). (E) Intracellular FFA are increased in immortalized AS podocytes when compared to immortalized WT podocytes (n = 3, * p < 0.05, t-test). (F,G) Mitochondrial area (F) and cristae density (G) of the podocytes in the TEM images were quantified by image J 1.54f. (* p < 0.05, **** p < 0.01, t-test). (H) Mitochondrial membrane potential (MMP) measured by aggregate/monomeric JC-1 ratio, demonstrating decreased MMP (n = 3, *** p < 0.001, **** p < 0.0001 one-way ANOVA). (I) ATP production was measured by bioluminescence (n = 3, ** p < 0.01 t-test). (J) Citrate synthase activity determined by the rate of absorbance gain at 412 nm normalized to protein concentration (n = 3, *** p < 0.001 t-test).

Figure 2

Ezetimibe improves LD–mitochondria contact formation and protects from immortalized podocyte injury. (A,B) TEM analysis demonstrating increased LD–mitochondria contact in immortalized AS podocytes treated with ezetimibe compared to untreated AS podocytes; red arrows point to LD and mitochondria contact, and blue arrows indicate the distance between LD and mitochondria (A), and the quantification (B) of LD and mitochondria contact formation (n = 3 mice per group, *** p < 0.001 **** p < 0.0001, one-way ANOVA). (C) The intracellular FFA quantification assay demonstrates that ezetimibe treatment reduces FFA in immortalized AS podocytes compared to untreated AS podocytes (n = 3, * p < 0.05, ** p < 0.01) (D) The apoptosis assay demonstrates that ezetimibe treatment of immortalized AS podocytes reduces apoptosis compared to untreated AS podocytes (n = 3–4, * p < 0.05, *** p < 0.001, one-way ANOVA). (E) The quantified mitochondrial area of the TEM image analysis demonstrates that ezetimibe treatment on immortalized AS podocytes prevents mitochondria swelling (n = 3, * p < 0.05, *** p < 0.001). (F,G) TEM image of Col4a3KO mice and ezetimibe-treated Col4a3KO mice, indicating that the ezetimibe treatment reduces the distance of LD and mitochondria (F) and the bar graph of the quantification (G) (n = 3, * p < 0.05).