The authors have declared that no competing interests exist.
Conceived and designed the experiments: XC XYB. Performed the experiments: JC SS SC QH. Analyzed the data: JC. Contributed reagents/materials/analysis tools: XS GC. Wrote the paper: JC XYB. Read and approved the final manuscript: JC SS XS GC SC QH XC XYB.
A high-calorie (HC) diet induces renal injury and promotes aging, and calorie restriction (CR) may ameliorate these responses. However, the effects of long-term HC and CR on renal damage and aging have been not fully determined. Autophagy plays a crucial role in removing protein aggregates and damaged organelles to maintain intracellular homeostasis and function. The role of autophagy in HC-induced renal damage is unknown.
We evaluated the expression of LC3/Atg8 as a marker of the autophagosome; p62/SQSTM1; polyubiquitin aggregates as markers of autophagy flux; Ambra1, PINK1, Parkin and Bnip3 as markers of mitophagy; 8-hydroxydeoxyguanosine (8-OHdG) as a marker of DNA oxidative damage; and p16 as a marker of organ aging by western blot and immunohistochemical staining in the kidneys of 24-month-old Fischer 344 rats. We also observed mitochondrial structure and autolysosomes by transmission electron microscopy.
Expression of the autophagosome formation marker LC3/Atg8 and markers of mitochondrial autophagy (mitophagy) were markedly decreased in the kidneys of the HC group, and markedly increased in CR kidneys. p62/SQSTM1 and polyubiquitin aggregates increased in HC kidneys, and decreased in CR kidneys. Transmission electron microscopy demonstrated that HC kidneys showed severe abnormal mitochondrial morphology with fewer autolysosomes, while CR kidneys exhibited normal mitochondrial morphology with numerous autolysosomes. The level of 8-hydroxydeoxyguanosine was increased in HC kidneys and decreased in CR kidneys. Markers of aging, such as p16 and senescence-associated-galactosidase, were increased significantly in the HC group and decreased significantly in the CR group.
The study firstly suggests that HC diet inhibits renal autophagy and aggravates renal oxidative damage and aging, while CR enhances renal autophagy and ameliorates oxidative damage and aging in the kidneys.
Diet has been long recognized as a modulator of kidney health in both humans and experimental models
In contrast, feeding mice with a high-calorie diet results in age-related obesity, cardiovascular diseases, and other metabolic disorders, and it shortens lifespan
In the past decade, more and more scientists explored the mechanism of the effect of HC and CR on aging
Autophagy is an evolutionarily conserved process in eukaryotic organisms. Cytoplasmic constituents are sequestered in double-membrane structures to form autophagosomes, which fuse with lysosomes to form autolysosomes. The cytoplasmic components are degraded by acid hydrolases, and the degradation products are released into the cytosol and recycled into biological structures or to supply energy during periods of starvation
Recent studies have revealed that both chronic (∼ 4 to 5 months) and short-term (4 or 8 weeks) high-fat diets can inhibit autophagy in the hypothalamus, skeletal and cardiac muscle. Other studies have demonstrated that 8–20 weeks of a high-fat diet can induce autophagy in pancreatic β-cells
In the present study, for the first time, we investigated the effect of a long-term (20-month) HC diet or CR on mitochondrial ultrastructure and the expression of LC3/Atg8 (as a marker of the autophagosome); p62/SQSTM1 and polyubiquitin aggregates (as markers of autophagy flux); Ambra1, PINK1, Parkin and Bnip3 (as markers of mitophagy); 8-hydroxydeoxyguanosine (8-OHdG) (as a marker of DNA oxidative damage); and p16 (as a marker of organ aging) in the kidneys of 24-month-old male Fischer 344 rats.
Male 3-month-old Fischer 344 rats were purchased. After adaptive feeding for one month in Experimental Animal Center of Chinese PLA General Hospital, these rats were randomly divided into three groups, and respectively given different diet interventions for 20 months. Control rats (n = 20) were fed a normal rat chow of 3.42 kcal/g (Keao, Beijing, China). HC rats (n = 25) were fed a modified chow of 4.51 kcal/g (Keao, Beijing, China). CR rats (n = 16) were fed 70% of the calorie intake of control rats. All animals were maintained in individual plastic cages and had free access to water. All experiments involving animals were approved by the Institutional Animal Care and Use Committee at the Chinese PLA General Hospital. Urine was collected in a tube using metabolic cages over a 24-h period and stored at −80°C.
At 24 months of age, six rats per group were anesthetized via intraperitoneal injection of sodium pentobarbital (40 mg/kg). The abdomen was opened and blood was collected from the abdominal aorta. Kidney tissues were removed and perfused with ice-cold, isotonic phosphate-buffered saline (PBS; pH 7.4) to remove any remaining blood. A portion of the renal tissue was fixed in 2.5% glutaraldehyde for transmission electron microscopy. The remaining tissues were immediately frozen in liquid nitrogen and stored at –80°C until further processing. The numbers of CON, CR and HC rats at the age of 3-month and 24-month and the mortality during the experiment were shown in
Group | 3-month-old | 24-month-old | Mortality during the 20 month | ||
CON | 20 | 14 | 6 | ||
CR | 16 | 14 | 2 | ||
HC | 25 | 8 | 17 |
CON, control animals; CR, calorie-restricted diet; HC, high-calorie diet.
CON | CR | HC | |
BMI (kg/m2) | 9.18±0.74 | 7.60±0.33 | 7.50±1.95 |
Kidney weight (g) | 2.73±0.18 | 2.95±0.37 | 3.70±0.18* |
Serum glucose (mmol/L) | 6.63±1.924 | 6.31±0.92 | 5.24±1.56 |
Serum creatinine (µmol/L) | 33.12±3.63 | 27.67±3.12 | 42.8±11.0* |
Serum triglyceride (mmol/L) | 2.56±0.85 | 0.53±0.18* | 3.56±1.36 |
Serum cholesterol (mmol/L) | 4.42±0.42 | 2.74±0.49* | 18.57±4.35* |
Urine protein/creatinine ratio (mg/mmol) | 529.35±139.9 | 256.37±98.38* | 1178.53±149.34* |
BMI, body mass index; CON, control animals; CR, calorie-restricted diet; HC, high-calorie diet. Data are presented as means ± SD (n = 6), *
Kidneys from the rats were excised, fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 4 µm for histological staining with periodic acid-Schiff followed by examination under the microscope.
The tissues from the cortex of the kidneys were lysed in RIPA buffer (50 mM Tris-Cl [pH 7.6], 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid, 1 µg/mL leupeptin, 1 µg/mL aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride) for 30 min on ice prior to centrifugation at 12,000 rpm for 30 min at 4°C. The protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis using a 6–15% acrylamide resolving gel and transferred to nitrocellulose membranes. Membranes were blocked in TBS-T (0.1% Tween-20) containing 5% milk for 1 h at room temperature followed by incubation with primary antibody at 4°C overnight. A rabbit anti-LC3 polyclonal antibody (1∶2000; Sigma, St. Louis, MO, USA), mouse monoclonal anti-p16 (1∶200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-ubiquitin (1∶2000; MABtech, Nacka Strand, Sweden), rabbit polyclonal anti-Parkin (1∶200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-PINK1 (1∶200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and mouse monoclonal anti-p62 (1∶200; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-Bnip3 (1∶5000; Abcam, Cambridge, MA, USA), rabbit polyclonal anti-Ambra1 (1∶5000; Abcam, Cambridge, MA, USA), or β-actin antibody (1∶5000; Sigma, St. Louis, MO, USA) was used to detect the corresponding proteins. Blots were subsequently probed with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG (Beyotime Institution of Biotechnology, Beijing, China) at 1∶1000–5000 dilutions. Immunoreactive bands were visualized by enhanced chemiluminescence and densitometry was performed using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
The kidneys were fixed in 10% formaldehyde overnight at 4°C and processed for paraffin-embedding following standard procedures. Sections were cut at 3-µm thicknesses. For immunohistochemical analysis, some tissue sections were subjected to antigen retrieval by microwaving or autoclaving for 10 or 15 min in 10 mM sodium citrate buffer [pH 6.0]. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 10 min. After PBS washing, sections were incubated with 1.5% normal goat serum for 20 min, followed by incubation with mouse monoclonal anti-8-OHdG antibody (1∶50; Santa Cruz Biotechnology Inc., CA, USA) and rabbit polyclonal anti-LC3 (1∶50; Sigma, St. Louis, MO, USA) overnight at 4°C. After three washes with PBS, the samples were incubated with biotin-conjugated goat anti-mouse IgG (Invitrogen Corporation, CA, USA) for 30 min at room temperature. After washing in PBS, the sections were incubated with streptavidin-conjugated peroxidase (Invitrogen Corporation, CA, USA) 30 min at room temperature. After PBS washing, the sections were incubated with DAB (Invitrogen Corporation, CA, USA) followed by examination under the microscope.
Four-micrometer kidney sections were fixed in 2% formaldehyde/0.2% glutaraldehyde at room temperature for 15 min. The sections were washed twice in PBS and incubated in freshly prepared SA-β-gal staining solution (1 mg/ml X-gal, 40 mM citric acid/sodium phosphate [pH 6.0], 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2) overnight at 37°C without CO2. Tissue sections were counterstained with eosin and examined under the light microscopy.
Kidneys were cut into tissue blocks (1 mm3) and fixed in 2.5% glutaraldehyde in 0.01 mol/L phosphate buffer at 4°C, followed by 2% osmium tetroxide. They were then dehydrated in a series of graded ethanol solutions. Ethanol was then substituted with propylene oxide and the tissue was embedded in epoxy resin. Ultrathin sections were double-stained with uranyl acetate and lead and examined under a JEM1200EX transmission electron microscope (JOEL) at 80 kV.
Analyses of all data were performed using the SPSS ver. 13.0 (SPSS, Chicago, IL, USA) software. Data are expressed as means ± standard deviation (SD). Comparisons among groups were made using analysis of variance. Values of
We analyzed changes in the metabolic parameters and renal functions in the control, HC and CR rats (
Periodic acid-Schiff (PAS) staining results showed that there were more obvious tubulointerstitial damages as well as a moderate inflammatory response in HC group compared to the CON group (
CON, control animals; CR, calorie-restricted diet; HC, high-calorie diet. Magnification, ×400.
The microtubule-associated protein 1 light chain 3 (LC3) is required for the formation of autophagosomes, especially the expansion of early autophagosomes
LC3 expression was analysed by Western blotting in the renal tissues of the CON, CR and HC Fischer 344 rats.
Autophagic flux refers to the complete process of autophagy including formation of autophagosomes, fusion of autophagosomes with lysosomes and their subsequent breakdown
The expression of p62/SQSTM1 and polyubiquitin aggregates was analyzed by Western blotting in kidneys from CON, CR, and HC Fischer 344 rats.
PTEN-induced kinase 1 (PINK1) and Parkin are mutated in many cases of early onset familial Parkinson’s disease (PD). Recent work suggests that PINK1 and Parkin function in the same pathway to maintain mitochondrial integrity
The expression of Parkin and PINK1 was analyzed by Western blotting in the kidneys of CON, CR, and HC Fischer 344 rats.
Bnip3 is anchored in the outer mitochondrial membrane via its C-terminal transmembrane domain (TMD), while the N-terminus faces the cytosol. The C-terminal TMD is essential for targeting Bnip3 to the mitochondria, homodimerization and pro-apoptotic activity. Interestingly, the N-terminus of Bnip3 contains a WXXL-like motif that might be important in binding to Atg8-family proteins
The expression of Bnip3 and Ambra1was analyzed by Western blotting in the kidneys of CON, CR, and HC Fischer 344 rats.
Ambra1 (activating molecule in Beclin 1-regulated autophagy) interacts with Parkin, a protein that promotes autophagy in the vertebrate central nervous system. Ambra1 is recruited in a Parkin-dependent manner to perinuclear clusters of depolarized mitochondria and contributes to their selective autophagic clearance
Oxidative stress, including reactive oxygen species (ROS), can damage both cellular macromolecules such as DNA, proteins, and lipids, and mitochondrial structure. In this study, we further observed the effect of diet on mitochondrial structures and the number of autolysosomes using transmission electron microscopy. The results showed obvious injury to mitochondrial structures, such as swelling and disintegration of cristae in the 24-month-old CON groups. There was also more severe mitochondrial damage and fewer autolysosomes in 24-month-old HC rats, whereas the 24-month-old CR groups exhibited relatively mild damage and more autolysosomes (
Analysis of mitochondrial structures and autolysosomes by transmission electron microscopy (TM) in the renal tissues of CON, CR, and HC Fischer 344 rats. White arrows indicate damagedmitochondria; black arrows indicate autolysosomes.CON, control animals; CR, calorie-restricted diet; HC, high-calorie diet.
Immunohistochemistry staining results for LC3 proteins and 8-OHdG in the kidneys of the CON, CR and HC Fischer 344 rats were scanned by a microscope. CON, control animals; CR, calorie-restricted diet; HC, high-calorie diet.
p16 is a robust biomarker and a possible effector in mammalian renal aging
The expression of the senescence biomarkers p16 and senescence-associated-galactosidase wasanalyzed in the kidneys of CON, CR, and HC Fischer 344 rats.
Previous study has shown that tubulointerstitial cell proliferation and interstitial accumulation of various extracellular matrix proteins progressively increases with increasing age in Milan rats
Compared with the above studies, in the present study, we mainly observed the effects of long-term diet (from 3-month-old to 24-month-old) intervention with either HC diet or CR on autophagy (especially mitophagy), oxidative damage and aging in the kidneys (mainly kidney tubular epithelial cells) of aged rats (24-month-old). We found that long-term HC markedly inhibited renal autophagy function, and that CR intervention may significantly increase the level of autophagy in the aged rat kidneys.
Eukaryotic cells have two major degradation systems, the autophagy–lysosome and proteasome pathways. Proteasomal degradation has high selectivity; the proteasome generally recognizes only ubiquitinated substrates, which are primarily short-lived proteins
In multicellular organisms, an important function of autophagy is the clearance of damaged or aged proteins or organelles, such as mitochondria. Recent studies suggest that this degradative process is selective and mediated by the mammalian protein p62/sequestosome 1 (SQSTM1)
Mitochondrial autophagy (mitophagy) is the only intracellular degradative mechanism for the removal of damaged mitochondria. Mitophagy was proposed to decrease the potential oxidative damage from defective mitochondria
Bnip3 promotes translocation of Parkin to the damaged mitochondria from the cytoplasm
Mitochondria are a major intracellular source of reactive oxygen species (ROS), and mitophagy is the only intracellular degradative mechanism for the removal of damaged mitochondria. Therefore, if mitophagy function is impaired and damaged mitochondria are not removed promptly, they will produce more ROS and further aggravate injury and aging of tissues and organs. In this study, we found that an HC diet results in more severe mitochondrial damage and increased levels of 8-OHdG and p16, while CR may ameliorate the mitochondrial damage and decrease the levels of 8-OHdG and p16 in the kidneys. This suggested that the excess energy intake from a HC diet might cause oxidative damage and aging via inhibition of mitochondrial autophagy in the kidneys, whereas restriction of caloric intake could increase mitochondrial autophagy and mitigate oxidative damage and aging in the kidneys.
Previous animal studies have shown that endoplasmic reticulum (ER) stress or oxidative stress induces early phase adaptive autophagy upregulation, which helps maintain intracellular homeostasis by disposing of a number of harmful molecules, such as cytosolic proteins damaged by ROS, and mitochondria
Studies in recent years have showed that deficiency and dysfunction in autophagy are implicated in the pathogenesis and progression of some renal diseases, such as glomerulosclerosis, renal ischemia/reperfusion injury, cyclosporine nephrotoxicity, and cisplatin nephrotoxicity
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