Excessive fat intake contributes to the progression of metabolic diseases via cellular injury and inflammation, a process termed lipotoxicity. Here, we investigated the role of lysosomal dysfunction and impaired autophagic flux in the pathogenesis of lipotoxicity in the kidney. In mice, a high-fat diet (HFD) resulted in an accumulation of phospholipids in enlarged lysosomes within kidney proximal tubular cells (PTCs). In isolated PTCs treated with palmitic acid, autophagic degradation activity progressively stagnated in association with impaired lysosomal acidification and excessive lipid accumulation. Pulse-chase experiments revealed that the accumulated lipids originated from cellular membranes. In mice with induced PTC-specific ablation of autophagy, PTCs of HFD-mice exhibited greater accumulation of ubiquitin-positive protein aggregates normally removed by autophagy than did PTCs of mice fed a normal diet. Furthermore, HFD-mice had no capacity to augment autophagic activity upon another pathologic stress. Autophagy ablation also exaggerated HFD-induced mitochondrial dysfunction and inflammasome activation. Moreover, renal ischemia-reperfusion induced greater injury in HFD-mice than in mice fed a normal diet, and ablation of autophagy further exacerbated this effect. Finally, we detected similarly enhanced phospholipid accumulation in enlarged lysosomes and impaired autophagic flux in the kidneys of obese patients compared with nonobese patients. These findings provide key insights regarding the pathophysiology of lipotoxicity in the kidney and clues to a novel treatment for obesity-related kidney diseases.

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Autophagy, an evolutionarily conserved cytoplasmic degradation system, has been implicated as a convergent mechanism in various longevity pathways. Autophagic activity decreases with age in several organisms, but the underlying mechanism is unclear. Here, we show that the expression of Rubicon, a negative regulator of autophagy, increases in aged worm, fly and mouse tissues at transcript and/or protein levels, suggesting that an age-dependent increase in Rubicon impairs autophagy over time, and thereby curtails animal healthspan. Consistent with this idea, knockdown of Rubicon extends worm and fly lifespan and ameliorates several age-associated phenotypes. Tissue-specific experiments reveal that Rubicon knockdown in neurons has the greatest effect on lifespan. Rubicon knockout mice exhibits reductions in interstitial fibrosis in kidney and reduced α-synuclein accumulation in the brain. Rubicon is suppressed in several long-lived worms and calorie restricted mice. Taken together, our results suggest that suppression of autophagic activity by Rubicon is one of signatures of aging.

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Autophagy plays an essential role in cellular homeostasis through the quality control of proteins and organelles. Although a time-dependent decline in autophagic activity is believed to be involved in the aging process, the issue remains controversial. We previously demonstrated that autophagy maintains proximal tubular cell homeostasis and protects against kidney injury. Here, we extend that study and examine how autophagy is involved in kidney aging. Unexpectedly, the basal autophagic activity was higher in the aged kidney than that in young kidney; short-term cessation of autophagy in tamoxifeninducible proximal tubule-specific autophagy-deficient mice increased the accumulation of SQSTM1/p62-and ubiquitin-positive aggregates in the aged kidney. By contrast, autophagic flux in response to metabolic stress was blunted with aging, as demonstrated by the observation that transgenic mice expressing a green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3B fusion construct, showed a drastic increase of GFP-positive puncta in response to starvation in young mice compared to a slight increase observed in aged mice. Finally, proximal tubule-specific autophagy-deficient mice at 24 mo of age exhibited a significant deterioration in kidney function and fibrosis concomitant with mitochondrial dysfunction as well as mitochondrial DNA abnormalities and nuclear DNA damage, all of which are hallmark characteristics of cellular senescence. These results suggest that age-dependent high basal autophagy plays a crucial role in counteracting kidney aging through mitochondrial quality control. Furthermore, a reduced capacity for upregulation of autophagic flux in response to metabolic stress may be associated with age-related kidney diseases.