Mitochondrial oxidative damage is a basic mechanism of aging, and multiple studies demonstrate that this process is attenuated by calorie restriction (CR). However, the molecular mechanism that underlies the beneficial effect of CR on mitochondrial dysfunction is unclear. Here, we investigated in mice the mechanisms underlying CR-mediated protection against hypoxia in aged kidney, with a special focus on the role of the NAD-dependent deacetylase sirtuin 1 (Sirt1), which is linked to CR-related longevity in model organisms, on mitochondrial autophagy. Adult-onset and long-term CR in mice promoted increased Sirt1 expression in aged kidney and attenuated hypoxia-associated mitochondrial and renal damage by enhancing BCL2/adenovirus E1B 19-kDa interacting protein 3-dependent (Bnip3-dependent) autophagy. Culture of primary renal proximal tubular cells (PTCs) in serum from CR mice promoted Sirt1-mediated forkhead box O3 (Foxo3) deacetylation. This activity was essential for expression of Bnip3 and p27Kip1 and for subsequent autophagy and cell survival of PTCs under hypoxia. Furthermore, the kidneys of aged Sirt1 +/-mice were resistant to CR-mediated improvement in the accumulation of damaged mitochondria under hypoxia. These data highlight the role of the Sirt1-Foxo3 axis in cellular adaptation to hypoxia, delineate a molecular mechanism of the CR-mediated antiaging effect, and could potentially direct the design of new therapies for age-and hypoxia-related tissue damage. IntroductionIncreasing age causes progressive postmaturational deterioration of tissues and organs, leading to impairment of tissue functioning, increased vulnerability to challenges, and death. The kidney is a typical target organ of age-associated tissue damage, and the increased incidence of chronic kidney disease (CKD) in the elderly is a health problem worldwide (1-3). However, there is little or no information on the mechanisms underlying age-associated kidney damage. Thus, studies designed to determine such molecular mechanisms could help formulate interventions that delay the onset and/or progression of CKD in elderly patients.Among the several proposed theories on the pathogenesis of ageassociated tissue damage, the mitochondrial ROS theory provides the basic mechanism of age-associated tissue dysfunction (4, 5): that age-dependent alteration in mitochondrial DNA (mtDNA) plays a fundamental role in the age-associated increase in ROS and subsequent tissue damage (6, 7). Further evidence linking alterations in mtDNA with progressive age-dependent tissue dysfunction can be found in individuals with mitochondrial genetic diseases and mice with deletion mutation of mtDNA, which display a phenotype that resembles premature aging, including kidney dysfunction (8,9). Hypoxia is the cause of age-associated mitochondrial dysfunction (10) and is involved in age-dependent tissue damage affecting the brain (11), heart (12), and kidney (13). Furthermore, hypoxia modulates various cellular processes, such as apoptosis, cell cycle, autophagy, and glucose me...

Metabolic syndrome has been reported to be associated with chronic kidney disease, but the mechanisms remain unclear. Although feeding of a high-fat diet (HFD) to C57BL/6 mice is reported to induce systemic metabolic abnormalities and subsequent renal injuries, such as albuminuria, similar to human metabolic syndrome, alterations in HFD-induced renal injuries have not been fully elucidated in detail. We therefore investigated the structural and functional changes in the kidneys of C57BL/6 mice on a HFD. Six-week-old mice were fed a low-fat diet (LFD; 10% of total calories from fat) or a HFD (60% fat) for 12 wk. Mice fed a HFD showed significant increases in body weight, systolic blood pressure, plasma insulin, glucose, and triglycerides compared with those on a LFD. Accompanying these systemic changes, mice on a HFD showed albuminuria, an increase in glomerular tuft area, and mesangial expansion. These systemic and renal alterations in mice on a HFD were prevented by body weight control with the dietary restriction of feeding a HFD. Furthermore, mice on a HFD showed renal pathophysiological alterations including renal lipid accumulation, an increased accumulation of type IV collagen in glomeruli, an increase in macrophage infiltration in the renal medulla, an increase in urinary 8-hydroxy-2'-deoxyguanosine excretion, and impaired sodium handling. In conclusion, this study suggests that local metabolic alterations in the kidney play important roles in the development of renal injury associated with metabolic syndrome in addition to systemic metabolic changes and an increase in body weight.

Overcoming refractory massive proteinuria remains a clinical and research issue in diabetic nephropathy. This study was designed to investigate the pathogenesis of massive proteinuria in diabetic nephropathy, with a special focus on podocyte autophagy, a system of intracellular degradation that maintains cell and organelle homeostasis, using human tissue samples and animal models. Insufficient podocyte autophagy was observed histologically in patients and rats with diabetes and massive proteinuria accompanied by podocyte loss, but not in those with no or minimal proteinuria. Podocyte-specific autophagy-deficient mice developed podocyte loss and massive proteinuria in a high-fat diet (HFD)-induced diabetic model for inducing minimal proteinuria. Interestingly, huge damaged lysosomes were found in the podocytes of diabetic rats with massive proteinuria and HFD-fed, podocyte-specific autophagy-deficient mice. Furthermore, stimulation of cultured podocytes with sera from patients and rats with diabetes and massive proteinuria impaired autophagy, resulting in lysosome dysfunction and apoptosis. These results suggest that autophagy plays a pivotal role in maintaining lysosome homeostasis in podocytes under diabetic conditions, and that its impairment is involved in the pathogenesis of podocyte loss, leading to massive proteinuria in diabetic nephropathy. These results may contribute to the development of a new therapeutic strategy for advanced diabetic nephropathy.

Metabolic syndrome is associated with increased risk of chronic kidney disease, and the renal injury in patients with metabolic syndrome may be a result of altered renal lipid metabolism. We fed wild-type or insulin-sensitive heterozygous peroxisome proliferator-activated receptor ␥-deficient (PPAR␥ ϩ/Ϫ ) mice a high-fat diet for 16 weeks. In wild-type mice, this diet induced core features of metabolic syndrome, subsequent renal lipid accumulation, and renal injury including glomerulosclerosis, interstitial fibrosis, and albuminuria. Renal lipogenesis accelerated, determined by increased renal mRNA expression of the lipogenic enzymes fatty acid synthase and acetyl-CoA carboxylase (ACC) and by increased ACC activity. In addition, renal lipolysis was suppressed, determined by reduced mRNA expression of the lipolytic enzyme carnitine palmitoyl acyl-CoA transferase 1 and by reduced activity of AMP-activated protein kinase. In PPAR␥ ϩ/Ϫ mice, renal injury, systemic metabolic abnormalities, renal accumulation of lipids, and the changes in renal lipid metabolism were attenuated. Thus, a high-fat diet leads to an altered balance between renal lipogenesis and lipolysis, subsequent renal accumulation of lipid, and renal injury. We suggest that renal lipid metabolism could serve as a new therapeutic target to prevent chronic kidney disease in patients with metabolic syndrome.

SIRT1, a class III histone deacetylase, is considered a key regulator of cell survival and apoptosis through its interaction with nuclear proteins. In this study, we have examined the likelihood and role of the interaction between SIRT1 and Smad7, which mediates transforming growth factor ␤ (TGF␤)-induced apoptosis in renal glomerular mesangial cells. Immunoprecipitation analysis revealed that SIRT1 directly interacts with the N terminus of Smad7. Furthermore, SIRT1 reversed acetyl-transferase (p300)-mediated acetylation of two lysine residues (Lys-64 and -70) on Smad7. In mesangial cells, the Smad7 expression level was reduced by SIRT1 overexpression and increased by SIRT1 knockdown. SIRT1-mediated deacetylation of Smad7 enhanced Smad ubiquitination regulatory factor 1 (Smurf1)-mediated ubiquitin proteasome degradation, which contributed to the low expression of Smad7 in SIRT1-overexpressing mesangial cells. Stimulation by TGF␤ or overexpression of Smad7 induced mesangial cell apoptosis, as assessed by morphological apoptotic changes (nuclear condensation) and biological apoptotic markers (cleavages of caspase3 and poly(ADP-ribose) polymerase). However, TGF␤ failed to induce apoptosis in Smad7 knockdown mesangial cells, indicating that Smad7 mainly mediates TGF␤-induced apoptosis of mesangial cells. Finally, SIRT1 overexpression attenuated both Smad7-and TGF␤-induced mesangial cell apoptosis, whereas SIRT1 knockdown enhanced this apoptosis. We have concluded that Smad7 is a new target molecule for SIRT1 and SIRT1 attenuates TGF␤-induced mesangial cell apoptosis through acceleration of Smad7 degradation. Our results suggest that up-regulation of SIRT1 deacetylase activity is a potentially useful therapeutic strategy for prevention of TGF␤-related kidney disease through its effect on cell survival.

Obesity is an independent risk factor for renal dysfunction in patients with CKDs, including diabetic nephropathy, but the mechanism underlying this connection remains unclear. Autophagy is an intracellular degradation system that maintains intracellular homeostasis by removing damaged proteins and organelles, and autophagy insufficiency is associated with the pathogenesis of obesity-related diseases. We therefore examined the role of autophagy in obesity-mediated exacerbation of proteinuria-induced proximal tubular epithelial cell damage in mice and in human renal biopsy specimens. In nonobese mice, overt proteinuria, induced by intraperitoneal free fatty acid-albumin overload, led to mild tubular damage and apoptosis, and activated autophagy in proximal tubules reabsorbing urinary albumin. In contrast, dietinduced obesity suppressed proteinuria-induced autophagy and exacerbated proteinuria-induced tubular cell damage. Proximal tubule-specific autophagy-deficient mice, resulting from an Atg5 gene deletion, subjected to intraperitoneal free fatty acid-albumin overload developed severe proteinuria-induced tubular damage, suggesting that proteinuria-induced autophagy is renoprotective. Mammalian target of rapamycin (mTOR), a potent suppressor of autophagy, was activated in proximal tubules of obese mice, and treatment with an mTOR inhibitor ameliorated obesity-mediated autophagy insufficiency. Furthermore, both mTOR hyperactivation and autophagy suppression were observed in tubular cells of specimens obtained from obese patients with proteinuria. Thus, in addition to enhancing the understanding of obesity-related cell vulnerability in the kidneys, these results suggest that restoring the renoprotective action of autophagy in proximal tubules may improve renal outcomes in obese patients.

Highlights d Energy metabolism shifts from lipolysis to ketolysis in damaged kidneys d mTORC1 hyperactivation leads to impaired renal lipolysis and subsequent renal damage d Ketone body supplementation ameliorates renal damage by blocking mTORC1 signaling d SGLT2 inhibitor-mediated renoprotection involves mTORC1 inhibition by ketone bodies

Oxidative stress is implicated to play an important role in the development of diabetic vascular complications, including diabetic nephropathy. It is unclear whether oxidative stress is primarily enhanced in the diabetic glomeruli or whether it is merely a consequence of diabetes-induced glomerular injury. To address this issue, we examined diabetic glomeruli to determine whether oxidative stress is enhanced, as well as examined the role of protein kinase C (PKC)-␤ activation in modulating NADPH oxidase activity. Urinary 8-hydroxydeoxyguanosine excretion and its intense immune-reactive staining in the glomeruli were markedly higher in diabetic than in control rats, and these alterations were ameliorated by a treatment with a selective PKC-␤ inhibitor, ruboxistaurin (RBX; LY333531) mesylate, without affecting glycemia. NADPH oxidase activity, which was significantly enhanced in diabetic glomeruli and the source of reactive oxygen species (ROS) generation, was also improved by RBX treatment by preventing the membranous translocation of p47phox and p67phox from cytoplasmic fraction without affecting their protein levels. Adenoviral-mediated PKC-␤ 2 overexpression enhanced ROS generation by modulating the membranous translocation of p47phox and p67phox in cultured mesangial cells. We now demonstrate that oxidative stress is primarily enhanced in the diabetic glomeruli due to a PKC-␤-dependent activation of NADPH oxidase resulting in ROS generation.