Methionine Restriction Delay Renal Senescence And Senescence Associated Secretory Phenotypes Via Endogenous Hydrogen Sulfide

Objective: Aging is an inevitable physiological process. Kidney has lots of changes with aging, such as reduced renal cortex, glomerular sclerosis, interstitial fibrosis, tubular atrophy and renal arteriosclerosis. Traditionally, cellular senescence has been considered a state of proliferative arrest, having cell-autonomous tumor suppressive functions in cancer and resulting in reduced tissue regenerative capacity during ageing. However, increasing attention has been focused on the non-cell-autonomous activities of senescent cells on the tissue microenvironment,which occur primarily through the senescence-associated secretory phenotype (SASP).The SASP reinforces senescence and activates an immune surveillance response, but it can also show pro-tumorigenic properties and contribute to age-related pathologies.Dietary restriction (DR) has been proved to be an effective way to delay aging. Many mechanisms contribute to this effect like increased autophagy, reduced inflammation and oxidative stress, increased insulin sensitivity and up-regulated SIRT1. At the same time, reducing the intake of specific nutrients, rather than the overall calorie, also plays a key role for the benefit. Especially the protein or specific amino acids restriction plays a more prominent role. Methionine restriction (MR) in the diet can protect the body by improving the metabolic markers, limiting fat accumulation and enhancing the insulin sensitivity. Previous studies have shown that MR can prolong the life span of animal models such as drosophila and mice. But the specific molecular mechanism remains unclear. Other studies have shown that endogenous hydrogen sulfide (H2S) production is essential for dietary restriction benefits. Sulfur amino acids such as methionine are precisely the important sources of H2S synthesis in vivo. Therefore, we speculated that MR can delay aging and prolong life span through the production of endogenous H2S.In the present study, we investigated the senescence-related markers (p16, p21 and p53) and SASP (IL-1?, IL-6, IL-8) in the kidney of 20-month-old male C57 mice which separated into Ad libitum (AL) and Methionine restriction (MR). We focused on whether methionine restriction in diet could delay renal aging by increasing the production of endogenous H2S. And we measured the level of pathway AMPK/mTOR to investigate the effect of H2S on this pathway. This study is of great significance in exploring new therapeutic regimens for delaying and curing the senescence-related renal diseases.Methods: Twelve male C57BL/6 mice were randomly divided into 2 groups: old methionine restriction group (OMR): methionine was limited to 0.15%, cystine was deficient in the diet (n=6), the old control group (old ad libitum, AL): free diet (n=6).Six 2-month-old male C57BL/6 mice were used as young control group (YAL). The other conditions were the same and the unit weight of the feed contained the same total calories. Food intake and weight change was monitored weekly. After 2 months, the mice were sacrifice. The tissues of the kidneys from each group of mice were removed.The biochemical indicators were measured. Western blot were performed to access the expressions of H2S synthase CGL, senescence markers p16, p21, p53 and the expression of AMPK / mTOR pathway which is related to senescence-associated secretory phenotype in renal tissue. The levels of IL-I? and IL-6 in the renal tissue homogenate were measured by enzyme-linked immunosorbent assay (ELISA).In vitro, the aging of human renal tubular epithelial cells (HK-2) was induced by indoxyl sulfate (IS), and the cells were divided into 3 groups: control group (DMEM),IS group (DMEM+250 ?M IS), IS+MR group (250 ?M IS + DMEM with 6mg/L methionine, lack of cysteine). HK-2 cells and culture supernatant were collect after 48 hours stimulation. Western blot were performed to access the expressions of H2S synthase CGL, senescence markers p16, p21, p53 and the expression of AMPK / mTOR pathway which is related to senescence-associated secretory phenotype in cell homogenate. The levels of IL-1 and IL-6 in culture supernatant were measured by enzyme-linked immunosorbent assay (ELISA). IL-1 and IL-8 were detected by immunofluorescence.In order to verify the key role of AMPK / mTOR pathway, AMPK inhibitor Compound C was added to the medium. Cells and culture supernatant were collect after 48 hours stimulation. Western blot and ELISA were performed to measure the corresponding protein.Result: 1) In vivo, OAL group showed obvious glomerular sclerosis,tubulointerstitial injury, renal tubular atrophy and vacuolar degeneration, inflammatory cell infiltration and other pathological changes, while the OMR group changed lighter,indicating that MR can delay the senescence-related renal changes. Western blot showed that compared with YAL group, the CGL in the kidney of OAL group was significantly decreased. And the senescence markers p16, p53, p21 were significantly increased.Phospho-AMPK prominently decreased. The phosphorylation level of mTOR was significantly increased, as well as phosphor-4E-BP1 which is the downstream of mTOR.But in OMR, the level of every indicate protein were different from OAL. 2) ELISA showed that the levels of IL-1? and IL-6 in the blood of the MR group were higher than those of the control group. 3) There were no significant differences related biochemical indicators between the two groups, but the blood glucose and cholesterol of MR group was significantly lower than that of AL group. 4) In vitro, compared with the control group, the expression of p53 and p21 in the IS group increased as well as the SA?-galactosidase positive cells, which confirmed the induction effect of IS on aging.Western blot showed that IS + MR group had significantly higher levels of CGL,phospho-AMPK and lower levels of p21, p53, phosphor-mTOR and phosphor-4E-BP1.5) ELISA showed that the concentration of H2S in IS + MR group was significantly increased, and the senescence-related secretory phenotypes of IL-1? and IL-6 in the culture supernatant were significantly decreased. 7) Immunofluorescence showed that IL-1? and IL-8 in IS + MR group decreased. 6) After the AMPK inhibitor Compound C was added, the expression of H2S and CGL were increased, while AMPK / mTOR,IL-1?, IL-6 in IS + MR group had no significant difference with those in IS group. The protection of MR weakened, which indicate H2S may regulate AMPK.Conclusion: The aging kidney is associated with increased senescence markers and SASP components (IL-1?, IL-6 and IL-8). Meanwhile, AMPK / mTOR pathway plays an important role in the regulation of SASP. The methionine restriction diet could inhibit the phosphorylation of mTOR, 4E-BP-1, the production of SASP and slow kidney aging by increasing the production of endogenous H2S and AMPK signaling pathway. Our study is of great significance in exploring new therapeutic regimens for delaying the senescence-related renal diseases.