| Objective:In the 21st century, aging of the population and the associated medical, social and economic implications have become one of the unneglectable issues. Under natural condition, aging is characterized by the progressive accumulation of damaged and defective cellular components, which results in a decline in the physiological function of tissues, loss of effected cells and causes organic aging. Autophagy is a highly regulated intracellular process for the degradation of cytoplasmic components, especially protein aggregates and damaged organelles. It is essential for maintaining healthy cells. Impaired or deficient autophagy is believed to cause or contribute to aging and age-related disease. Calorie restriction (CR) is the only intervention to date that has consistently been shown to slow the rate of aging and to increase mean and maximum lifespan in a variety of species. Interestingly, autophagy and lysosomal proteolysis can be stimulated in virtually all cells by depressed levels of serum amino acids, raising the possibility that CR stimulates autophagy. In this study, we investigated the effects of age on autophagy in the kidneys of 3-,12-, and 24-month-old Fischer 344 rats. Meanwhile, we investigated the effects of diet on macroautophagy in the kidneys of 24-month-old Fischer 344 rats that were fed either ad libitum (CON), a CR diet or a high calorie diet (HG).Methods:3-month-old 63 male Fischer 344 rats were randomly divided into three groups:control group, calorie restriction(CR) group, high-calorie(HG) group. Control group:free diet; calorie restriction group:limit to 70% of normal food intake (food intake to control rats a week as the standard changes in food intake, at any time); high-calorie group:free to eat high-calorie diet. Food intake was monitored daily and the weight change of the rats was detected monthly. Male Fischer 344 rats at the ages of 3,12,24 months were used for this study, as well as the kidney of 24-month-old Fischer 344 rats that were fed either ad libitum (CON), a calorie restriction diet or a high calorie diet (HG). The renal tissues were processed for senescence analyses. Renal senescence was detected by (3-galactosidae staining. Atg5 and Atg7 levels were quantified by real-time PCR. Western blot analysis was performed to assess p16, LC3, beclin-1, p62/SQSTM1 and polyubiquitin aggregates. Expression and location of LC3 and 8-OHdG in kidneys were studied by immunofluorescence staining. The changes of mitoch--ondria were analyzed by transmission electron microscope. Paraffin-embedded renal tissues were prepared to do PAS staining. We also examined the content of malondialdehyde (MDA), protein carbonyl.Results:SA-β-gal staining was seen in tubular which frequency and intensity was increased with the increasing of age. The protein expression levels of pi6, p62/SQSTM1, polyubiquitin aggregates were increased significantly in 24-month-rat kidneys. The mRNA and protein expression levels of Atg7 and LC3 in rat kidneys were decreased markedly with advancing age. Analysis by transmission electron microscope demonstrated swelling and disintegration of cristae in the mitochondria of aged kidneys. The 8-OHdG staining by immunofluorescence and immunohistochemistry staining were increased in 24-month-rat kidneys. The levels of 8-OHdG, p16, beclin-1, p62/SQSTMl and polyubiquitin aggregates were significantly elevated in HG group than CR or CON groups. The level of Atg5 was decreased obviously in CR group than HG or CON groups. The level of LC3 was increased in CR group than HG or CON group. Determination of protein carbonyl and MDA were found, compared with the control group, protein carbonyl groups were increased in HG group, there is significant difference (P<0.05), but in CR group protein carbonyl groups were significantly lower (P<0.05).Conclusions:These results suggest that autophagy function decreases with age in the kidneys of Fischer 344 rats, and autophagy may mediate the process of kidney aging, leading to the accumulation of damaged mitochondria. We propose that an overall stimulation of the autophagy response caused by calorie restriction, as we have demonstrated for rat kidney tissue, will sustain the removal of damaged cellular components and may thereby enhance the survival of renal cells. |
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