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The Effects of Age and Different Calorie Diet on the Function of Autophagy in Rat Kidneys

Author: CuiJing
Tutor: BaiXueYuan
School: PLA Postgraduate Medical School
Course: Within the scientific kidney disease
Keywords: Senescence Autophagy Oxidative damage Kidney Mitochondria
CLC: R692
Type: Master's thesis
Year: 2011
Downloads: 86
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Abstract

Objective: At present, China has entered the aging society, the aging problem has become one of the major issues facing the state and society. Therefore, the urgent need to deepen the understanding of the mechanism of aging in the kidney and to explore strategies to delay the aging kidney, thereby reducing the occurrence and development of senile kidney disease. Currently considered aging essentially damaged and defective cellular components continue to accumulate in the cells, eventually causing the decline of physical function of cells and tissues, resulting in the aging body. Autophagy (autophagy), that self-the phagocytosis (autophagocytosis) exists in a highly conserved eukaryotic cells to maintain intracellular environment important protective mechanism. Under physiological conditions, autophagy can remove a small amount of damage to intracellular organelles and macromolecular aggregates when autophagy injured or autophagy defects, the body may be aging and aging-related diseases. Studies have shown that the energy is closely related to the development of metabolic abnormalities with aging. Calorie restriction has been recognized only delay the aging rate of multiple species, to extend the intervention of the average and maximum life. However, the molecular mechanism of anti-aging calorie restriction is not very clear. This study by observing the change of 3,12 and 24-month-old Fischer 344 rat kidney autophagy, clear aging of autophagy functions of the kidneys; explore the high-calorie diet and calorie restriction on aging rat kidney autophagy function, has important implications for the understanding of the occurrence and development mechanism of the aging kidney and kidney disease, as well as exploring new treatment options for anti-aging-related kidney disease. Methods: (1) take the 3-month-old, 12-month-old and 24-month-old rats (n = 10), kidney tissue, detection of renal pathological changes in rats with PAS stain. Observation autophagy-related genes (autophagy related genes, of Atg) LC3/Atg8 of beclin-1/Atg6, Atg5, Atg7 lysosomal cathepsin B and ubiquitin-binding protein p62/SQSTM1 and real time-PCR and Western blot, polyubiquitin aggregates (polyubiquitin aggregates) expression change. Immunohistochemical method observed DNA damage product 8 - hydroxy-deoxyguanosine (8-OHdG) in renal tissue positioning and expression. Immunofluorescence assay of 8-OHdG and LC3 localization and expression changes in kidney tissue. Respectively with the senescence-associated β-galactosidase galactosidase activity (SA-β-gal) staining method and Western blot detection of SA-β-gal activity changes and aging signs protein of p16. Kidney mitochondria structural changes detected by transmission electron microscopy. (2) 3-month-old male Fischer 344 rats 63 were randomly divided into three groups: ① normal diet group: 26, freedom of drinking water; ② calorie restriction group: 16, limited to the normal food intake of 70%; ③ The high-calorie diet group: 21, free to eat high-calorie diet. Until the rats were fed to 24 months of age were sacrificed and specimens from the 24-hour urine, and blood. Detect blood biochemistry, renal function (Scr, BUN), and urine protein / creatinine. Measure the body weight of rats when the rats were killed, length, kidney weight. The OCT-embedded and 10% formalin-fixed specimens from kidney tissue stored in liquid nitrogen. Respectively for the detection of p16 protein and SA-β-gal, since changes in the macrophage-associated protein Atg, lipid and protein oxidative damage malondialdehyde (MDA) and protein carbonyl, 8-OHdG, PAS staining rat renal pathology changed. Results: We first observe a the kidney function autophagy change in the aging process. ① estern blot, compared with 3-month-old and 12-month-old rats, 24-month-old rat kidney senescence markers p16 expression levels significantly and the aging of β-galactosidase activity of phase with the 3-month-old, 12-month-old The ratio increased significantly. The ② estern blot display, compared to 3-month-old rat kidney tissue, 24-month-old rats autophagy markers LC3-Ⅰ with increasing age significantly reduced with increasing age, LC3-II did not change significantly. Immunofluorescence results showed that 24-month-old rat kidney LC3 expression significantly reduced. The ③ quantitative PCR results showed that Atg7 expression in rat kidney with increasing age significantly reduce. ④ estern blot results show that the ubiquitin-conjugating aggregates polyubiqintin aggregates protein p62/SQSTM1 and polyubiquitin rat kidney tissue at 24 months of age was significantly higher with increasing age. Beclin-1/Atg6 no significant changes with increasing age. Cathepsin B expression was significantly reduced in the kidneys of 24-month-old rats. ⑤ immunofluorescence and immunohistochemistry results showed that, in the 12-month-old and 24-month-old rat kidney DNA damage product 8-OHdG expression was significantly higher than the 3-month-old rats. ⑥ TEM observations showed that: 12-month-old and 24-month-old rat kidney tissue, renal tubular epithelial cells, mitochondria swelling deformation and structure of mitochondrial cristae disorders. ⑦ kidney tissue PAS staining showed: compared with the 3-month-old, 24-month-old rats glomerulosclerosis. Seen tubulointerstitial inflammatory cell infiltration, tubular atrophy. We further explore the impact of different calorie diet intervention on the kidney function of autophagy. ① Compared with the normal diet group, a high-calorie diet group rat kidney, p16 expression was significantly increased, and significantly lower in the calorie restriction group. ② normal diet rats compared to kidney tissue, high-calorie diet group rat kidney tissue LC3-Ⅰ and LC3-Ⅱ significantly reduce calorie restriction renal tissue LC3-Ⅰ and LC3-Ⅱ was significantly higher. The immunofluorescent results show calorie restricted rats kidney tissue LC3 expression than normal diet group, a high-calorie diet group LC3 expression is lower than the normal diet group. ③ Compared with the normal diet group rat kidney on, p62/SQSTM1 polyubiqintin aggregates expressed in rat kidney tissue in the calorie restriction group was significantly lower, and the high-calorie diet group was significantly higher. ④ immunofluorescence results show that, compared with the normal diet group, a high-calorie diet group of 8-OHdG expression increased caloric restriction reduced expression of the group of 8-OHdG. ⑤ oxidative damage markers carbonyl and malondialdehyde (MDA) detection, high-calorie diet group was significantly higher than the normal diet group, calorie restriction group was significantly lower than the normal diet group. (6) in renal tissue PAS staining showed that: compared with the normal diet group, a high-calorie diet group mesangial cell proliferation and in renal interstitial inflammation cells infiltration increased, the degree of calorie restriction group mesangial cell proliferation and tubular interstitial inflammatory cell infiltration lighter. Conclusion: With increasing age autophagy in rat kidney function decline, accompanied by the increase in oxidative damage and aging. That impaired autophagy may play an important role in the occurrence and development of kidney aging. Calorie restriction can enhance the function of the kidney tissue of aged rats autophagy, weakened kidney oxidative damage and anti-aging; contrary, high-calorie diet can inhibit autophagy function of the kidney tissue of aged rats, increased kidney oxidative damage and accelerated aging .

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