LIPID Metabolism Dysfunction–Induced LYSOSOMAL FRAGILITY DISRUPTS AUTOPHAGY AND PROMOTES ACUTE KIDNEY INJURY PROGRESSION

Acute kidney injury (AKI) is a major risk factor for chronic kidney disease and mortality, with injury to proximal tubular epithelial cells (PTECs) playing a central role in its pathogenesis. Autophagy in PTECs is essential for maintaining cellular homeostasis and responding to stress, and it has been shown to promote recovery from AKI. While impaired autophagy has been reported in AKI, the underlying mechanisms responsible for this dysfunction remain unclear. Meanwhile, lipid accumulation is frequently observed in PTECs during AKI. However, it remains uncertain whether this reflects a mere metabolic abnormality or actively contributes to tubular injury. In this study, we focused on how disrupted lipid metabolism affects autophagic activity, particularly through its relationship with lysosomal function, in an effort to elucidate a novel molecular mechanism underlying the progression of AKI.

We employed murine models of ischemia-reperfusion (I/R) and cisplatin-induced AKI, as well as cultured PTECs exposed to hypoxia-reoxygenation (H/R). Autophagic flux was assessed using GFP-LC3 mice and mRFP-GFP-LC3 reporters. Lysosomal structure and function were evaluated by electron microscopy, DQ-BSA degradation, cathepsin B/D activity, lysotracker staining, and galectin-3 puncta for lysosomal membrane permeabilization (LMP). Lipid peroxidation was quantified by untargeted lipidomics and 4-hydroxynonenal (4-HNE) staining. Interventions included PGC1α overexpression, PPARα agonist fenofibrate, ferrostatin-1 (a lipid peroxidation inhibitor), and Ppara knockout. Key findings were validated using human kidney biopsy samples from ischemic AKI patients.

PTECs exhibited marked autophagic flux impairment during AKI, evidenced by LC3-II and p62 accumulation and reduced autolysosome formation. Lysosomes appeared swollen, exhibited reduced acidification, and showed diminished cathepsin activity. Galectin-3 puncta and cytosolic cathepsin leakage indicated LMP, while electron microscopy confirmed lysosomal structural damage. These changes coincided with significant lipid droplet accumulation and elevated 4-HNE. In vitro, exogenous 4-HNE recapitulated LMP and autophagic blockade. Enhancing fatty acid oxidation via PGC1α or fenofibrate restored mitochondrial ATP levels, reduced 4-HNE, preserved lysosomal integrity, and reactivated autophagy. Ferrostatin-1 effectively suppressed lipid peroxidation, protected lysosomal structure and proteolytic function, and attenuated kidney injury. Conversely, Ppara-deficient mice showed exacerbated 4-HNE accumulation, intensified LMP, and worsened AKI. Human ischemic AKI samples recapitulated key features observed in mouse models, including lysosomal swelling, galectin-3 positivity, and elevated lipid peroxidation in PTECs.

Our findings define a pathogenic lipid–lysosome–autophagy axis that drives AKI progression. Oxidative lipid stress impairs lysosomal function via LMP, arresting autophagic degradation and promoting tubular injury. Restoring lipid metabolic homeostasis or inhibiting lipid peroxidation preserves lysosomal integrity and offers a promising therapeutic strategy. The consistent pathological signatures across models and human tissue underscore the translational relevance of targeting lysosomal fragility and oxidative lipid damage in AKI.