Salt-Responsive Gut Microbiota Induces Sex-Specific Blood Pressure Changes

Abstract

Background: Tryptophan metabolism is important in blood pressure regulation. The tryptophan-indole pathway is exclusively mediated by the gut microbiota. ACE2 (angiotensin-converting enzyme 2) participates in tryptophan absorption, and a lack of ACE2 leads to changes in the gut microbiota. The gut microbiota has been recognized as a regulator of blood pressure. Furthermore, there is ample evidence for sex differences in the gut microbiota. However, it is unclear whether such sex differences impact blood pressure differentially through the tryptophan-indole pathway.

Methods: To study the sex-specific mechanisms of gut microbiota-mediated tryptophan-indole pathway in hypertension, we generated a novel rat model with Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9)-targeted deletion of Ace2 in the Dahl salt-sensitive rat. Cecal microbiota transfers from donors of both sexes to female S recipients were performed. Also, Dahl salt-sensitive rats of both sexes were orally gavaged with indole to investigate blood pressure response.

Results: The female gut microbiota and its tryptophan-indole pathway exhibited greater buffering capacity when exposed to tryptophan, due to Ace2 deficiency, and salt. In contrast, the male gut microbiota and its tryptophan-indole pathway were more vulnerable. Female rats with male cecal microbiota responded to salt with a higher blood pressure increase compared with those with female cecal microbiota. Indole, a tryptophan-derived metabolite produced by gut bacteria, increased blood pressure in male but not in female rats. Moreover, salt altered host-mediated tryptophan metabolism, characterized by reduced serum serotonin of both sexes and higher levels of kynurenine derivatives in the females.

Conclusions: We uncovered a novel sex-specific mechanism in the gut microbiota-mediated tryptophan-indole pathway in blood pressure regulation. Salt tipped the tryptophan metabolism between the host and gut microbiota in a sex-dependent manner. Our study provides evidence for a novel concept that gut microbiota and its metabolism play sex-specific roles in the development of salt-sensitive hypertension.

Keywords: blood pressure; gastrointestinal microbiome; hypertension; salts; sex characteristics.

Figure 1.

CRISPR/Cas9-mediated deletion of Ace2 locus increases blood pressure in both male and female S rats but female heterozygous Ace2^−/+ is protected from salt-induced hypertension. (A) This was obtained from https://www.sciencedirect.com/science/article/pii/S1043661823002761?via%3Dihub with permission (open access). A 935 base pairs deletion within the rat Ace2 locus that covers partial intron 1 and exon 2 was confirmed by amplification of genomic DNA from NF (left full band) and KO rat (right lower band). Reduced ACE2 protein activity in the serum of Ace2 deficient S rats of both (B) male and (C) female as compared with NF. NF male, n = 6, Ace2^−/y male, n=6; NF female, n = 4, Ace2^−/+ female, n=6, Ace2^−/− female, n=7. Data were analyzed by unpaired t test and one-way ANOVA (Kruskal-Wallis test). Systolic blood pressure (SBP) comparison between NF and Ace2 deficient S rats in male (D-G) and female (H-K) from baseline (low salt) to three weeks of high salt. NF male, n = 7, Ace2^−/y male, n=7; NF female, n = 7, Ace2^−/+ female, n=7, Ace2^−/− female, n=5. Data were analyzed by 2-way ANOVA. ****p<0.0001. P_time, P_genotype and P_interaction are presented in supplementary Table S3. Relative expression levels of inflammatory mediators in the ileum of NF and Ace2 deficient S rats on high salt in (L-N) male and (O-Q) female. NF male, n = 6, Ace2^−/y male, n=6; NF female, n = 7, Ace2^−/+ female, n=8, Ace2^−/− female, n=5. Data were analyzed by unpaired t test and ordinary one-way ANOVA.

Figure 1.

CRISPR/Cas9-mediated deletion of Ace2 locus increases blood pressure in both male and female S rats but female heterozygous Ace2^−/+ is protected from salt-induced hypertension. (A) This was obtained from https://www.sciencedirect.com/science/article/pii/S1043661823002761?via%3Dihub with permission (open access). A 935 base pairs deletion within the rat Ace2 locus that covers partial intron 1 and exon 2 was confirmed by amplification of genomic DNA from NF (left full band) and KO rat (right lower band). Reduced ACE2 protein activity in the serum of Ace2 deficient S rats of both (B) male and (C) female as compared with NF. NF male, n = 6, Ace2^−/y male, n=6; NF female, n = 4, Ace2^−/+ female, n=6, Ace2^−/− female, n=7. Data were analyzed by unpaired t test and one-way ANOVA (Kruskal-Wallis test). Systolic blood pressure (SBP) comparison between NF and Ace2 deficient S rats in male (D-G) and female (H-K) from baseline (low salt) to three weeks of high salt. NF male, n = 7, Ace2^−/y male, n=7; NF female, n = 7, Ace2^−/+ female, n=7, Ace2^−/− female, n=5. Data were analyzed by 2-way ANOVA. ****p<0.0001. P_time, P_genotype and P_interaction are presented in supplementary Table S3. Relative expression levels of inflammatory mediators in the ileum of NF and Ace2 deficient S rats on high salt in (L-N) male and (O-Q) female. NF male, n = 6, Ace2^−/y male, n=6; NF female, n = 7, Ace2^−/+ female, n=8, Ace2^−/− female, n=5. Data were analyzed by unpaired t test and ordinary one-way ANOVA.

Figure 2.

Ace2 deficiency induces changes in gut microbiota on low salt. Bacterial α diversity indices Chao1 richness and Shannon diversity comparison between NF rats and Ace2 deficient S rats in males (A, B) and females (E, F). Low salt male NF, n = 5, LS male Ace2^−/y, n = 6; LS female NF, n = 4, LS female Ace2^−/+, n = 6, LS female Ace2^−/−, n = 7. Data were analyzed by non-parametric Mann-Whitney test and one-way ANOVA (Kruskal–Wallis test). Principal coordinate analysis (β diversity, Bray-Curtis) comparison between NF rats and Ace2 deficient S rats in males (C) and females (G). The cluster ellipse shows the 95% confidence interval assuming a multivariate normal distribution. Bar plots representing the differentially abundant bacterial genera in NF (blue) and Ace2^−/y (red) in males (D). Data were analyzed by linear discriminant analysis effect size (LEfSe) with FDR Adjusted p-value <0.05, LDA>2.0.

Figure 3.

Ace2 deficiency induces more significant changes in male gut microbiota on high salt. Bacterial α diversity indices Chao1 richness and Shannon diversity comparison between NF rats and Ace2 deficient S rats in males (A, B) and females (E, F). High salt male NF, n = 7, male Ace2^−/y, n = 6; female NF, n = 7, female Ace2^−/+, n = 8, female Ace2^−/−, n = 5. Data were analyzed by unpaired t test and ordinary one-way ANOVA. *p<0.05. Principal coordinate analysis (β diversity, Bray-Curtis) comparison between NF rats and Ace2 deficient S rats in males (C) and females (G). The cluster ellipse shows the 95% confidence interval assuming a multivariate normal distribution. Bar plots representing the differentially abundant bacterial genera in NF (blue) and Ace2^−/y (red) in males (D). Data were analyzed by linear discriminant analysis effect size (LEfSe) with FDR Adjusted p-value <0.05, LDA>2.0.

Figure 4.

Ace2 deficiency increased indole levels mainly in males. (A-D) Cecal and serum tryptophan and indole levels in males of both genotypes (NF, Ace2^−/y). (E-H) Cecal and serum tryptophan and indole levels in females of the three genotypes (NF, Ace2^−/+, and Ace2^−/−). Low salt (LS) male NF, n = 5, LS male Ace2^−/y, n = 6; High salt (HS) male NF, n = 7, HS male Ace2^−/y, n = 6; LS female NF, n = 4, LS female Ace2^−/+, n = 6, LS female Ace2^−/−, n = 7; HS female NF, n = 7, HS female Ace2^−/+, n = 8, HS female Ace2^−/−, n = 5. Data were analyzed by non-parametric Mann-Whitney test. *p<0.01.

Figure 5.

Escherichia Shigella abundance is correlated with indole levels mainly in males. Mantel test correlation between serum and cecum indole levels with gut bacterial genus level abundance in S rats of all genotypes: (A) low salt, males, (B) low salt, females, (C) high salt, males, and (D) high salt, females. Relationship among bacterial genera was presented as Pearson’s correlation.

Figure 5.

Escherichia Shigella abundance is correlated with indole levels mainly in males. Mantel test correlation between serum and cecum indole levels with gut bacterial genus level abundance in S rats of all genotypes: (A) low salt, males, (B) low salt, females, (C) high salt, males, and (D) high salt, females. Relationship among bacterial genera was presented as Pearson’s correlation.

Figure 6.

Male cecal microbiota induces a greater blood pressure increase in female S rats in response to high salt. (A) Schematics of the experiment. (B) Female S rats were given either male or female cecal microbiota. Upon high salt diet, both groups experienced increased blood pressure, but the rise was greater in rats with male cecal microbiota. Data were presented as ΔSBP, which was calculated by the SBP difference between indicated days on high salt and day 0 baseline SBP on low salt. Female S rats with male cecal content, n=6; Female S rats with female cecal content, n=6. SBP, systolic blood pressure. Data were analyzed by two-way ANOVA. *p<0.05, **p<0.01, ns, not significant. P_time, P_treatment and P_interaction are presented in supplementary Table S3.

Figure 7.

Indole increases blood pressure in male, but not female, NF S rats. Male systolic blood pressure at baseline (A) and after 3 weeks of oral gavage with indole (B). Female systolic blood pressure at baseline (C) and after 3 weeks of oral gavage with indole (D). PBS-Control treated male, n = 6, Indole treated male, n = 9; PBS-Control treated female, n = 6, Indole treated female, n = 8. Data were analyzed by two-way ANOVA. ****p < 0.0001, ns, not significant. P_time, P_treatment and P_interaction are presented in supplementary Table S3.

Figure 8.

Salt altered host-mediated tryptophan metabolism. (A) HS decreased serum serotonin levels in both sexes. (B) Females had elevated levels of kynurenine compared to male S rats on both LS and HS. Elevated levels of kynurenine metabolites (kynurenic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid) in the serum of females compared with males, and HS amplified this difference (C-E). Low salt (LS) male, n = 6, High salt (HS) male, n = 7; LS female, n = 4, HS female, n = 7. Data were analyzed by non-parametric Mann-Whitney test. *p<0.01.