Alterations of the gut microbial community structure and function with aging in the spontaneously hypertensive stroke prone rat

Abstract

Gut dysbiosis, a pathological imbalance of bacteria, has been shown to contribute to the development of hypertension (HT), systemic- and neuro-inflammation, and blood-brain barrier (BBB) disruption in spontaneously hypertensive stroke prone rats (SHRSP). However, to date individual species that contribute to HT in the SHRSP model have not been identified. One potential reason, is that nearly all studies of the SHRSP gut microbiota have analyzed samples from rats with established HT. The goal of this study was to examine the SHRSP gut microbiota before, during, and after the onset of hypertension, and in normotensive WKY control rats over the same age range. We hypothesized that we could identify key microbes involved in the development of HT by comparing WKY and SHRSP microbiota during the pre-hypertensive state and longitudinally. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and fecal microbiota analyzed by16S rRNA gene sequencing. SHRSP showed significant elevations in SBP, as compared to WKY, beginning at 8 weeks of age (p < 0.05 at each time point). Bacterial community structure was significantly different between WKY and SHRSP as early as 4 weeks of age, and remained different throughout the study (p = 0.001-0.01). At the phylum level we observed significantly reduced Firmicutes and Deferribacterota, and elevated Bacteroidota, Verrucomicrobiota, and Proteobacteria, in pre-hypertensive SHRSP, as compared to WKY. At the genus level we identified 18 bacteria whose relative abundance was significantly different in SHRSP versus WKY at the pre-hypertensive ages of 4 or 6 weeks. In an attempt to further refine bacterial candidates that might contribute to the SHRSP phenotype, we compared the functional capacity of WKY versus SHRSP microbial communities. We identified significant differences in amino acid metabolism. Using untargeted metabolomics we found significant reductions in metabolites of the tryptophan-kynurenine pathway and increased indole metabolites in SHRSP versus WKY plasma. Overall, we provide further evidence that gut dysbiosis contributes to hypertension in the SHRSP model, and suggest for the first time the potential involvement of tryptophan metabolizing microbes.

Figure 1

Systolic blood pressure (SBP) in WKY and SHRSP from 6 to 20 weeks of age. Two-way repeated measures ANOVA showed statistical main effects of strain, age, and interaction between strain and age (p < 0.001 for all, n = 5–7). *p ≤ 0.002 compared to corresponding age in WKYs (Holm-Sidak method).

Figure 2

PCoAs of weighted UniFrac, measure of β diversities, comparing SHRSPs and WKYs at 4, 6, 8, 10, 16, and 20 weeks of age. The p-values, as determined using permutational multivariate analysis of variance (PERMANOVA), comparing SHRSPs and WKYs at each age group are presented at the top of each plot. n = 6–15.

Figure 3

Relative abundance of phyla in SHRSP and WKY at 4, 6, 8, 10, 16 and 20 weeks (n = 6–15). * and **p < 0.05 and 0.01 respectively compared to WKY at the same age using Mann–Whitney U test with FDR corrected for multiple comparisons.

Figure 4

Relative abundance of genera in SHSRP and WKY at 4, 6, 8, 10, 16 and 20 weeks (n = 6–15). * and ** p < 0.05 and 0.01 respectively compared to WKY at the same age by Mann–Whitney U test with FDR corrected for multiple comparisons.

Figure 5

Relative abundance of genera in WKY (top) and SHRSP (bottom) during aging from 4 to 20 weeks. (n = 6–15) *, **, and ***p < 0.05, 0.01, and 0.001 over age respectively using Mann–Whitney U test with FDR corrected for multiple comparisons. Only genera with relative abundance ≥ 0.5% are shown.

Figure 6

(A) Heatmap of Spearman correlations between gut-brain modules (GBM) and genera relative abundance. Genus-GBM pairs with correlations >  = 0.6 or <  = −0.6 and p-value < 0.05 are shown. (B) Short-chain fatty acid measurement of WKY and SHRSP plasma. (C) Schematic diagram of tryptophan metabolism pathways. D-E. WKY and SHRSP plasma metabolites of kynurenine pathway (D) and indole pathway (E). (n = 6) *p < 0.05 using Mann–Whitney U test with FDR corrected for multiple comparisons.