Differential effects of sodium chloride and monosodium glutamate on kidney of adult and aging mice

Abstract

Monosodium Glutamate (MSG) is used as flavour enhancer, with potential beneficial effects due to its nutritional value. Given the decline in kidney functions during aging, we investigated the impact of MSG voluntary intake on the kidney of male mice, aged 6 or 18 months. For 2 months, they freely consumed water (control group), sodium chloride (0.3% NaCl) or MSG (1% MSG) in addition to standard diet. Young animals consuming sodium chloride presented signs of proteinuria, hyperfiltration, enhanced expression and excretion of Aquaporin 2 and initial degenerative reactions suggestive of fibrosis, while MSG-consuming mice were similar to controls. In old mice, aging-related effects including proteinuria and increased renal corpuscle volume were observed in all groups. At an advanced age, MSG caused no adverse effects on the kidney compared to controls, despite the presence of a sodium moiety, similar to sodium chloride. These data show that prolonged MSG intake in mice has less impact on kidney compared to sodium chloride, that already in young animals induced some effects on kidney, possibly related to hypertension.

Figure 1

Albuminuria in male mice at the end of treatment. Albumin excretion was evaluated with silver staining. Urinary samples obtained from 6 and 18 months old mice, whose diet was supplemented for 2 months with water, sodium chloride or MSG (Ctrl, NaCl and MSG, respectively), were examined. N = 8 (young Ctrl), 7 (young NaCl), 8 (young MSG), 5 (old Ctrl), 6 (old NaCl), 7 (old MSG). Mann–Whitney test **p < 0.01.

Figure 2

Kidney weight and water/substance intake measurements. (A) Ratio of total kidney (left plus right) weight to body weight in 6 months (young) or 18 months (old) old mice groups. An increase in kidney weight was associated with aging in each treatment group (n = 21 in each group. t test: *p < 0.05, **p < 0.01, ***p < 0.001). (B, C) Water (B) and (C) substance (Water, NaCl and MSG respectively) consumption normalized by total mouse weight within a cage. (D) Body weight at the end of treatment. (E) Heart weight normalized to body weight (mg/g) at the end of treatment.

Figure 3

Effect of diet in association with age on renal phenotype. (A) Kidney PAS staining was performed on 5 male mice randomly selected within each group. Black arrow points to proteinaceous casts and black stars areas mark increased cellular proliferation, respectively. (B) Mean number of casts in each group (n = 5 mice/group), no casts were detected in young control and MSG groups. In old mice, NaCl versus MSG group Mann–Whitney U = 3.50, p = 0.056. (C, D) Bowman’s Capsule volume (C) and Glomerular volume (D) morphometric measures were performed in PAS-stained tissues. N = 150 glomeruli from 5 mice in each group. t test, *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Evidence of kidney fibrosis evaluated with histology staining, αSMA labelling and fibronectin deposition. (A) Representative Gomori trichrome renal staining (× 40). (B) The dot plot analysed the fibrosis intensity in function of age and treatment (n = 25 sections from 5 mice in each group, t test *p < 0.05, **p < 0.01). (C) Immunofluorescence showing the presence of αSMA in the kidney. The green channel, unmarked, display the background and shows the morphology of the tissue. Scale bar, 50 µm. The panels in the first row represent the 3D reconstruction of the bidimensional images (scale bar 20 µm). (D) Fibronectin deposition in the kidney, a representative Western blot is shown. (E) Analysis of fibronectin deposition, as a proportion of fibronectin normalized to GAPDH (A.U. arbitrary units, n = 3/group). No fibronectin was detected in young control and MSG mice.

Figure 5

Analysis of AQP2 excretion in male mice urine. (A) Urine was collected from mice supplemented with water (control, Ctrl), NaCl (NaCl) or MSG (MSG) at two different end point time (young and old, 6 and 18 months respectively). A typical immunoblot with urinary samples is shown. Two bands can be seen, the top being glycosylated and the bottom non-glycosylated AQP2. (B) Summary of quantitative data (n = 6 in Old NaCl group, 5 in all other groups) obtained from immunoblot analysis with relative statistical analysis (t test, **p < 0.01).

Figure 6

AQP2 expression and localization in kidney tissue. (A) Western blot analysis showing the effects of dietary supplementation and age on the expression of AQP2. (B) AQP2 relative intensity analysis performed on WB (A.U. arbitrary units, n = 5). Representative immunohistochemistry (C) and immunofluorescence (D) showing the tissue distribution of AQP2 in collecting ducts. The green channel, unmarked, displays the background and shows the morphology of the tissue. Scale bar: 50 µm. The panels in the first row represent the 3D reconstruction of AQP2 bidimensional image series (scale bar: 20 µm).