Time-dependent dysregulation of autophagy: Implications in aging and mitochondrial homeostasis in the kidney proximal tubule

<p>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 tamoxifen-inducible 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.</p

Lipophagy maintains energy homeostasis in the kidney proximal tubule during prolonged starvation

<p>Macroautophagy/autophagy is a self-degradation process that combats starvation. Lipids are the main energy source in kidney proximal tubular cells (PTCs). During starvation, PTCs increase fatty acid (FA) uptake, form intracellular lipid droplets (LDs), and hydrolyze them for use. The involvement of autophagy in lipid metabolism in the kidney remains largely unknown. Here, we investigated the autophagy-mediated regulation of renal lipid metabolism during prolonged starvation using PTC-specific <i>Atg5</i>-deficient (<i>atg5</i>-TSKO) mice and an in vitro serum starvation model. Twenty-four h of starvation comparably induced LD formation in the PTCs of control and <i>atg5</i>-TSKO mice; however, additional 24 h of starvation reduced the number of LDs in control mice, whereas increases were observed in <i>atg5</i>-TSKO mice. Autophagic degradation of LDs (lipophagy) in PTCs was demonstrated by electron microscopic observation and biochemical analysis. In vitro pulse-chase assays demonstrated that lipophagy mobilizes FAs from LDs to mitochondria during starvation, whereas impaired LD degradation in autophagy-deficient PTCs led to decreased ATP production and subsequent cell death. In contrast to the in vitro assay, despite impaired LD degradation, kidney ATP content was preserved in 48-h starved <i>atg5</i>-TSKO mice, probably due to increased utilization of ketone bodies. This compensatory mechanism was accompanied by a higher plasma FGF21 (fibroblast growth factor 21) level and its expression in the PTCs; however, this was not essential for the production of ketone bodies in the liver during prolonged starvation. In conclusion, lipophagy combats prolonged starvation in PTCs to avoid cellular energy depletion.</p

Antioxidant role of autophagy in maintaining the integrity of glomerular capillaries

<p>Autophagy is a lysosomal degradation system by which cytosolic materials and damaged organelles are broken down into basic components. To explore the physiological role of autophagy in glomerular endothelial cells (GEnCs), we compared the autophagic flux among cells in the kidney under starvation. Inhibition of autophagy by chloroquine administration significantly increased the number of autophagosomes or autolysosomes in GEnCs and proximal tubular cells, but not in podocytes, suggesting that the GEnCs exhibit substantial autophagic activity. Next, we analyzed endothelial and hematopoietic cell-specific <i>atg5</i>-deficient mice (<i>atg5</i>-conditional KO [cKO] mice). Glomeruli of 4-wk-old <i>atg5</i>-cKO mice exhibited slightly distended capillary loops accompanied by an accumulation of reactive oxygen species (ROS). Glomeruli of 8-wk-old <i>atg5</i>-cKO mice showed a lobular pattern with thickening of the capillary loops and mesangial matrix expansion; however, the vasculature of other organs was preserved. The <i>atg5</i>-cKO mice died by 12 wk of age, presumably due to pancytopenia resulting from the defect in their hematopoietic lineages. Therefore, we subjected 4-wk <i>atg5</i>-cKO mice to irradiation followed by bone marrow transplantation from normal littermates. Transplanted mice recapitulated the glomerular phenotypes of the <i>atg5</i>-cKO mice with no obvious histological changes in other organs. Twelve-mo-old transplanted mice developed mesangiolysis and glomerulosclerosis with significant deterioration of kidney function. Administration of N-acetyl-l-cysteine, a ROS scavenger, to <i>atg5</i>-cKO mice rescued the glomerular phenotypes. These data suggest that endothelial autophagy protects glomeruli from oxidative stress and maintains the integrity of glomerular capillaries. Enhancing endothelial autophagy may provide a novel therapeutic approach to minimizing glomerular diseases.</p