Methionine restriction delays senescence and suppresses the senescence-associated secretory phenotype in the kidney through endogenous hydrogen sulfide

Figures & data

Figure 1. Increased longevity in C57BL/6 mice fed a MR diet. Three-month-old C57BL/6 mice were randomized into a control group (n = 10) and a diet group (n = 10). The control group (AL) was fed AIN-93 chow, and the diet group (MR) was fed chow in which the methionine content was reduced to 15% of that in the control diet.

Table 1. Metabolic parameters and renal functions in the OAL and OMR groups

	OAL group	OMR group
Body weight (g)	32.15 ± 2.09	30.20 ± 1.24
Kidney weight/Body weight	0.0065 ± 0.00089	0.0069 ± 0.00123
Albumin (g/L)	33.53 ± 4.62	34.25 ± 2.26
Total protein (g/L)	57.5 ± 4.62	55.2 ± 2.62
Glucose (mmol/L)	11.36 ± 1.67	7.13 ± 0.48*
Creatinine (μmol/L)	14.36 ± 4.90	16.40 ± 8.67
Serum urea nitrogen (mmol/L)	7.46 ± 0.74	8.70 ± 1.47
Cholestrol (mmol/L)	3.61 ± 0.36	2.47 ± 0.36*
Triglycerides (mmol/L)	0.77 ± 0.03	0.71 ± 0.06
Glycosylated serum protein (μmol/L)	134.5 ± 28.3	129.9 ± 25.1
Ca (mmol/L)	2.4 ± 0.19	2.2 ± 0.14
P (mmol/L)	3.0 ± 0.52	3.3 ± 0.28

OAL, old ad libitum; OMR, methionine restriction. Data are presented as means±SD (n = 6–8).* p < 0.05 OMR vs. OAL.

Figure 2. Representative pictures and histology scores of glomerular and tubulointerstitial lesions based on standard procedures. (a) The black arrows indicate a fibrous crescent, cell proliferation, and glomerulosclerosis in the glomerular lesions and interstitial inflammatory cell infiltration in the tubulointerstitial lesions. MR improved glomerular and tubulointerstitial lesions. (magnification ×400) (b) Histology scores based on standard procedures were not significantly different between the OMR and OAL groups. (c-e) The levels of albuminuria, KIM-1, and NGAL in urine of different groups. The quantitative analysis included 20 random fields per mouse. The data are presented as the means ± SD (n = 6–8).

Figure 3. MR slows senescence and suppresses the SASP in the kidneys through TSP and AMPK/mTOR. (a and b) Levels of senescence markers p16, p53, p21 and IL-1β in the young group (3-month-old), control group (OAL), and diet group (OMR). (c and d) Levels of IL-1β and IL-6 in the kidneys in the young group (3-month-old), control group (OAL), and diet group (OMR). (magnification ×40) (e and f) MR increases the TSP key enzyme CGL and H2S levels. (e) Levels of the TSP key enzyme CGL in the kidneys in the young group (3-month-old), control group (OAL), and diet group (OMR). (f) Concentrations of H2S in the serum in the young group (3-month-old), control group (OAL), and diet group (OMR). *P < 0.05. (g-i) MR increases phospho-AMPK and attenuates phosphor-mTOR levels. (G) Levels of phospho-AMPK in the kidneys in the young group (3-month-old), control group (OAL), and diet group (OMR). (h-j) Levels of phospho-mTOR and its downstream target phospho-4EBP1 and phosphor-S6K in the kidneys. Three replicates were used for western blot quantification. Values are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus OAL. NS, not significant. (n = 6–8).

Figure 4. MR slows IS-induced senescence and suppresses the SASP in HK-2 cells. (a–c) Western blot analyses showing the levels of the senescence markers p53 and p21 in the three groups. (d) Serum-starved HK-2 cells were incubated with or without IS (250 μM) for 48 h; then, the cells were stained for SA β-gal, and the number of SA β-gal-positive cells was counted. (magnification ×400) (e and f) The levels of IL-1β and IL-6 in cell culture supernatants were analysed by ELISA. (g-i) Immunofluorescence results show IL-1β and IL-8 staining. The fluorescence intensity was lower in the IS+MR group than in the IS group. (magnification ×200). Three replicates were used for quantification. Values are presented as means ± SD. *P < 0.05, **P < 0.01.

Figure 5. MR increases H₂S production and AMPK/mTOR phosphorylation. (a) Levels of CGL assessed by Western blot; CGL activation was lower in the IS group than in the IS+MR group. (b) H₂S levels were increased by MR in HK-2 cells. (c and d) Phosphorylated AMPK levels were significantly increased after MR treatment. The protein band intensities are presented as ratios compared to the intensities of the total bands; the data from the control group were arbitrarily set as 1.0. Three replicates were used for quantification. Values are presented as means ± SD.

Figure 6. Suppression of IS-induced senescence in HK-2 cells by MR is regulated by H₂S and AMPK/mTOR. Cells were incubated with 8 μM CC and with or without IS for 48 h. (a and b) Levels of the senescence markers p53 and p21 were analysed by Western blot. (c and d) Levels of IL-1β and IL-6 in cell culture supernatants were analysed by ELISA. (e) Cellular H₂S concentrations. (f and g) Western blots showing changes in CGL and the AMPK/mTOR pathway. *p < 0.05，**p < 0.01，***p < 0.001 versus IS+MR. #p < 0.05 versus IS+MR.