Early and late gut microbiota signatures of stroke in high salt-fed stroke-prone spontaneously hypertensive rats

Abstract

The high salt-fed stroke-prone spontaneously hypertensive rat (SHRSP) is a suitable tool to study the mechanisms underlying stroke pathogenesis. Salt intake modifies the gut microbiota (GM) in rats and humans and alterations of the GM have previously been associated with increased stroke occurrence. We aimed to characterize the GM profile in SHRSPs fed a high-salt stroke-permissive diet (Japanese diet, JD), compared to the closely related stroke-resistant control (SHRSR), to identify possible changes associated with stroke occurrence. SHRSPs and SHRSRs were fed a regular diet or JD for 4 weeks (short-term, ST) or a maximum of 10 weeks (long-term, LT). Stroke occurred in SHRSPs on JD-LT, preceded by proteinuria and diarrhoea. The GM of JD-fed SHRSPs underwent early and late compositional changes compared to SHRSRs. An overrepresentation of Streptococcaceae and an underrepresentation of Lachnospiraceae were observed in SHRSPs JD-ST, while in SHRSPs JD-LT short-chain fatty acid producers, e.g. Lachnobacterium and Faecalibacterium, decreased and pathobionts such as Coriobacteriaceae and Desulfovibrio increased. Occludin gene expression behaved differently in SHRSPs and SHRSRs. Calprotectin levels were unchanged. In conclusion, the altered GM in JD-fed SHRSPs may be detrimental to gut homeostasis and contribute to stroke occurrence.

Keywords: Gut barrier; Gut microbiota; High-salt diet; SHRSP; Stroke.

Figure 1

Phenotypic parameters of SHRSRs and SHRSPs after short-term (ST, 4 weeks) regular diet (RD) or Japanese diet (JD). Body weight change (%) during 4 weeks of RD (a) or JD (b) feeding in the two rat strains. (c) Proteinuria levels after 4 weeks of RD or JD feeding in the two rat strains. (d) Systolic blood pressure levels at the same experimental times. Two-way ANOVA followed by Tukey post-hoc comparison. In panels (a) and (b), only different rat strains at the same time point were statistically compared. SHRSR RD, n = 8 in panel a, n = 9 in panel (c) and (d); SHRSP RD, n = 7; SHRSR JD-ST, n = 7; SHRSP JD-ST, n = 7. (e) Azan Masson’s trichrome staining showed a significant increase in perivascular (*) and glomerular ( <) fibrosis in SHRSPs, but not SHRSRs, fed JD for 4 weeks compared with the corresponding controls fed RD. Bar graphs show quantification of fibrosis as percentage of blue. Data are mean + /– standard error of the mean (SEM). Mann–Whitney two-tailed test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Phenotypic parameters of SHRSRs and SHRSPs under long-term Japanese diet (JD-LT) feeding. (a) Body weight change (%) during long-term JD feeding in the two rat strains. Two-way ANOVA followed by Tukey post-hoc comparison calculated only untill the first rat died of stroke (after 6 weeks). Only different rat strains at the same time point were statistically compared. SHRSR JD-LT, n = 11; SHRSP JD-LT, n = 13 up to week 5; SHRSP JD-LT, n = 10 at week 6; SHRSP JD-LT, n = 5 at week 7; SHRSP JD-LT, n = 3 at week 8; SHRSP JD-LT, n = 2 at week 9. (b) Proteinuria and (c) blood pressure levels after long-term JD feeding in the two rat strains. One-way ANOVA followed by Tukey post-hoc comparison. SHRSR JD-LT, n = 11 at all timepoints; SHRSP JD-LT, n = 13 at week 4; SHRSP JD-LT, n = 9 at week 6; SHRSP JD-LT, n = 3 at week 8. **p < 0.01, ***p < 0.001.

Figure 3

Stroke occurrence in SHRSRs and SHRSPs upon long-term JD feeding. Percentage of stroke events in the two rat strains upon JD-LT treatment. The comparison between the two strains was significantly different (Log-rank and Mann–Whitney test, p < 0.0001). For the number of animals included in this analysis, see Fig. 2.

Figure 4

Phylum and family-level gut microbiota composition of SHRSRs and SHRSPs after 4 weeks of regular diet (RD) or Japanese diet (JD-ST), and after long-term JD feeding (JD-LT). Bar plots showing the relative abundance of major phyla (a) and families (b) in the large intestine of the two rat strains after 4 weeks of RD or JD feeding (JD-ST) and after long-term JD feeding (JD-LT). Only taxa with a relative abundance > 0.1% in at least 3 samples are shown. SHRSR RD, n = 9; SHRSP RD, n = 7; SHRSR JD-ST, n = 8; SHRSP JD-ST, n = 7; SHRSR JD-LT, n = 11; SHRSP JD-LT, n = 13.

Figure 5

Family-level gut microbiota signatures of SHRSRs and SHRSPs after 4 weeks of regular diet (RD) or Japanese diet (JD-ST), and after long-term JD feeding (JD-LT), and correlations with blood pressure and proteinuria levels. (a) Heatmap showing Ward-linkage clustering based on Pearson's correlation coefficients of the relative abundance of gut microbiota families of the two rat strains after 4 weeks of RD or JD feeding (JD-ST) and after long-term JD feeding (JD-LT). Only taxa with a relative abundance > 0.1% in more than 2 samples are shown. See Supplementary Fig. S2 for further details. Scatter plots of correlation between relative abundance of families and (b) blood pressure (BPmmHG) and (c) proteinuria levels in SHRSRs and SHRSPs after RD or JD-ST/JD-LT. Only statistically significant correlations (p ≤ 0.05) based on Kendall rank correlation test with absolute tau ≥ 0.2 are shown.

Figure 6

Calprotectin concentration in the serum of SHRSRs and SHRSPs after 4 weeks of regular diet (RD) or Japanese diet (JD-ST), and after long-term JD feeding (JD-LT). Values in ng/ml (mean ± SD) are shown for the two rat strains after 4 weeks of RD or JD feeding (JD-ST) and after long-term JD feeding (JD-LT). SHRSR RD, n = 7; SHRSP RD, n = 5; SHRSR JD-ST, n = 7; SHRSP JD-ST, n = 6; SHRSR JD-LT, n = 10; SHRSP JD-LT, n = 13.

Figure 7

Relative gene expression of ZO-1 and Ocln in the small intestinal mucosa of SHRSRs and SHRSPs after 4 weeks of regular diet (RD) or Japanese diet (JD-ST), and after long-term JD feeding (JD-LT). Relative expression was calculated as fold change with respect to SHRSR RD or SHRSP RD groups. The error bar represents the range of relative expression. *p < 0.05, **p < 0.01, ***p < 0.001, (a–d) t-test; (e–f) two-way ANOVA, post-hoc standard with Tukey correction. SHRSR RD, n = 9; SHRSP RD, n = 7; SHRSR JD-ST, n = 8; SHRSP JD-ST, n = 7; SHRSR JD-LT, n = 11; SHRSP JD-LT, n = 13.

Figure 8

Diagram of experimental groups and samplings. SHRSR and SHRSP animals were sacrificed after 4 weeks of regular diet (RD) (SHRSR RD and SHRSP RD groups, respectively), or after 4 weeks (short term, ST) of Japanese Diet (JD) (SHRSR JD-ST and SHRSP JD-ST groups, respectively). JD-fed rats continued JD (long-term, LT) for 10 weeks before being sacrificed (SHRSR JD-LT group) or until death by stroke, which occurred between week 6 and week 9 (SHRSP JD-LT group). Serum, large and small intestines, and kidneys were collected after sacrifice. Body weight of each animal was measured weekly from the beginning of the diet until sacrifice, and blood pressure and proteinuria were measured during the 4th, 6th, 8th and 10th week of the diet. Stroke onset was continuously monitored throughout the experiment. The figure was created using icons from https://www.freepik.com/.