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Bile acids (BA) are classically viewed as emulsifiers produced by the liver for aiding the absorption of fats in the gut. Accumulating evidence now recognizes BA as vasoactive agents capable of lowering blood pressure (BP). However, it remains unclear whether the ensuing hypotension can be associated with end-organ damage. To delineate the pathogenic role of BA in the gut-liver-kidney axis, we leveraged our prior finding that a subset of C57BL/6 mice developed cholemia ( i.e. , high circulating BA) when fed a diet supplemented with 7.5% inulin. Briefly, we challenged 4-week-old male mice (n=50) with the inulin diet for one week and identified the subset that developed cholemia (B6 BA ; n=6). Non-cholemic mice (B6; n=6) were maintained as control. Assessment on BP at 6 months of inulin feeding indicated that B6 BA mice (systolic BP: 107.6 ± 2.2 mmHg; diastolic BP: 78.6 ± 3.1 mmHg) are hypotensive relative to non-cholemic B6 mice (systolic BP: 139.8 ± 5.6 mmHg; diastolic BP: 109.0 ± 3.8 mmHg), which was accompanied by polydipsia (7.0 ± 0.4 ml/day in B6 BA vs 4.3 ± 0.2 ml/day water intake in B6), polyuria (2.5 ± 0.1 ml/day in B6 BA vs 1.2 ± 0.1 ml/day urine output in B6), and elevated urinary BA (28.5 ± 1.7 μM in B6 BA vs 3.3 ± 0.6 μM in B6) in B6 BA mice. B6 BA mice also excreted more urinary creatinine (50.4 ± 3.4 mg/dl in B6 BA vs 25.6 ± 1.3 mg/dl in B6), sodium (271.8 ± 20.1 μmol/day in B6 BA vs 103.0 ± 11.0 μmol/day in B6) and potassium (349.2 ± 16.7 μmol/day in B6 BA vs 128.7 ± 7.7 μmol/day in B6), which could be, in part, explained by their higher glomerular filtration rate (1209.0 ± 121.0 in B6 BA vs 681.2 ± 148.7 μl/min/100 g body weight in B6). Of note, B6 BA mice displayed renomegaly, interstitial nephritis, and bile casts in urine, as well as elevated renal transcripts (>2-fold increase relative to B6) for lipocalin 2, kidney injury molecule-1, tumor necrosis factor alpha, collagen type I α1 and endothelin-1. Taken together, these findings demonstrate that our mouse model of diet-induced cholemia presents with low BP and renal pathology. We envision that this model has notable advantages ( e.g. , ease in inducing cholemia, no early morbidity and tractable for long-term study), in lieu of the conventional bile duct ligation approach, for studying how BA regulate BP.

Neutrophils are important innate immune cells that are relatively understudied for their contributions to blood pressure (BP) regulation. As neutrophilia is a clinical marker for various pathologies, we hypothesized that neutrophilia is also a feature of hypertension and curtailing neutrophilia is beneficial for lowering BP. Peripheral neutrophil levels were quantitated in Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats consuming either a low salt (0.3% NaCl) or high salt (2% NaCl) diet. Compared to R rats, S rats on a low-salt diet already exhibited neutrophilia (% neutrophils, 33.51±1.6 vs. 39.74±1.2, pi.e., generation of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs, web-like structures of expelled neutrophil DNA). Peripheral neutrophils stimulated with either PMA or LPS showed a ~ 3-fold induction of ROS and NETs, respectively, in S rats compared to R rats on a low-salt diet. Introducing a high-salt diet increased ROS and NETs (~2-fold), the extent of elevation being higher in S rats. Impressively, treatment of S rats on a high-salt diet with 1,3-butandiol [(β - hydroxybutyrate (βOHB, a ketone body) precursor] in the drinking water (20% v/v, 4 weeks) significantly lowered neutrophilia (vehicle: 1.74±0.5 vs βOHB: 0.73±0.3 x 10 9 cells/l, p9 cells/l), albeit to a lesser degree than S rats. Importantly, βOHB administration to S and R rats also reduced NETs and ROS (~2-fold), predominantly in S rats. Collectively, our data are the first to demonstrate that an exaggerated innate immune response via neutrophilia is pathological in hypertension, whereby curbing the neutrophilic response ( e.g., βOHB) could present therapeutic potential in lowering BP.

Commensal gut microbiota are strongly correlated with host hemodynamic homeostasis, but only broadly associated with cardiovascular health. This includes a general correspondence of quantitative and qualitative shifts in intestinal microbial communities found in hypertensive rat models and human patients. However, the mechanisms by which gut microbes contribute to the function of organs important for blood pressure control remain unanswered. To examine the direct effects of microbiota on blood pressure, we conventionalized germ-free (GF) rats with specific pathogen free rats for a short-term period of 10 days, which served as a model system to observe the dynamic responses when reconstituting the holobiome. The absence of microbiota in GF rats resulted with relative hypotension compared to their conventionalized counterparts, suggesting an obligatory role of microbiota in blood pressure homeostasis. Hypotension observed in GF rats was accompanied by a marked reduction in vascular contractility. Both blood pressure and vascular contractility were restored by the introduction of microbiota to GF rats, indicating that microbiota could be impacting blood pressure through a vascular-dependent mechanism. This is further supported by the decrease in actin polymerization in arteries from GF rats. Improved vascular contractility in conventionalized GF rats, as indicated through stabilized actin filaments, was associated with an increase in cofilin phosphorylation. These data indicate that the vascular system senses the presence (or lack of) microbiota to maintain vascular tone via actin polymerization. Taken together, these results constitute a fundamental discovery of the essential nature of microbiota in blood pressure regulation.

Alterations of diurnal rhythms of blood pressure (BP) and reshaping of gut microbiota are both independently associated with hypertension. However, the relationships between biorhythms of BP and gut microbial composition are unknown. We hypothesized that diurnal timing-associated alterations of microbial compositions are synchronous with diurnal rhythmicity, dip in BP, and renal function. To test this hypothesis, Dahl salt-sensitive (S) rats on low- and high-salt diets were examined for time of day effects on gut microbiota, BP, and indicators of renal damage. Major shifts in night and day patterns of specific groups of microbiota were observed between the dark (active) and light (rest) phases, which correlated with diurnal rhythmicity of BP. The diurnal abundance of Firmicutes, Bacteroidetes, and Actinobacteria were independently associated with BP. Discrete bacterial taxa were observed to correlate independently or interactively with one or more of the following 3 factors: (1) BP rhythm, (2) dietary salt, and (3) dip in BP. Phylogenetic Investigation of Communities revealed diurnal timing effects on microbial pathways, characterized by upregulated biosynthetic processes during the active phase of host, and upregulated degradation pathways of metabolites in the resting phase. Additional metagenomics functional pathways with rhythm variations were noted for aromatic amino acid metabolism and taurine metabolism. These diurnal timing dependent changes in microbiota, their functional pathways, and BP dip were associated with concerted effects of the levels of renal lipocalin 2 and kidney injury molecule-1 expression. These data provide evidence for a firm and concerted diurnal timing effects of BP, renal damage, and select microbial communities.

Circadian, diurnal rhythm is a vital physiological feature of life forms, which enables holobionts to adapt to the day and night cycles. Evidence suggests that both factions of the holobiont, i.e, the host and its microbiota demonstrate physiological circadian rhythms. Blood pressure is a good example of a host physiological feature with a well-defined diurnal rhythm. In a healthy human, blood pressure (BP) rises to its peak during awakening morning hours and declines to the lowest level during night. In salt-sensitive hypertension, aberrant diurnal rhythms of BP and gut dysbiosis have been demonstrated. Given the critical role of gut microbiota in BP regulation, our current objective was to investigate whether there are synchronous rhythms of holobiont in rodents on low salt and high salt diets and if inflammation pattern also changes diurnally. We examined Dahl Salt-Sensitive (S) rats on low (0.3%) and high (2%) salt diets and BP and inflammation pattern was checked. As hypothesized, both microbiota and kidney inflammation showed diurnal rhythm in response to low salt and high salt diet. Major shifts in diurnal patterns of specific groups of microbiota were observed between the dark (active) and light (rest) phases, which correlated with the diurnal rhythmicity of BP. Diurnal rhythms of Firmicutes, Bacteroidetes and Actinobacteria were independently associated with BP. Discrete bacterial taxa were observed to correlate independently or interactively with one or more of the following 3 factors- 1) BP rhythm, 2) dietary salt, 3) amplitude of BP. PICRUSt analysis revealed diurnal rhythmicity of microbial pathways, characterized by microbiota upregulated biosynthetic processes during active phase and upregulated degradation pathways of metabolites in resting phase. These diurnal changes in microbiota, their functional pathways and BP amplitude were associated with concerted rhythmicity of renal Lipocalin 2 and Kim1 expression and circulating β-hydroxybutyrate in high salt S rats. Such concerted rhythmicity of holobiont with peak of changes at active phase of salt hypertension suggests that targeting this timepoint to reshape microbiota and/or intervene with medication could efficiently benefit the hypertensives

Elevated blood pressure or hypertension is the single largest risk factor for cardiovascular diseases which are the leading cause of human deaths. Current clinical management of blood pressure is focused on restoring homeostasis of the host alone, without accounting for commensal gut microbiota. Recent evidence from the CARDIA study in humans and multiple studies using animal models suggest that development of hypertension in the host is associated with alterations in microbiotal communities. Here we examined whether microbiota is necessary for blood pressure and vascular homeostasis by functional evaluation of the gut homeostasis, hemodynamic, and vascular function of gnotobiotic rats reconstituted with microbiota to represent the complete holobiont. Gnotobiotic rats were used to represent incomplete holobionts. To reconstitute complete holobionts, gnotobiotic rats were co-housed with conventionally-raised rats. Acquisition of microbiota was evaluated through monitoring of gross ceca and fecal samples by metagenomic 16S sequencing. BP was recorded and vascular, renal, hepatic, cardiac and gut features were assessed using histology and ex vivo myography. Markers of innate immune effectors (Immune cell population, level of Lcn2, Gut permeability) were used to examine the nature and extent of host immune cell processes concomitantly occurring along with observations of host hemodynamics. Compared to the reconstituted holobiont represented by the animals exposed to microbiota, the incomplete-holobiont represented by gnotobiotic rats, had significantly lower BP (SBP of germ free:109±8 mmHg, SBP of conventionalized:138±10mmHg * ) and vascular contractility responses to phenylephrine (Emax (mN): germ-free: 6.9±1.3, GFC: 11.7±0.7*). Acute exposure of the host to microbiota reconstituted gut microbiotal communities, significantly boosted their gut epithelial cell proliferation, innate immune function and restored vascular contractility. These data indicate that in addition to the dependency of the host on microbiota for essential bodily functions such as digestion of plant-derived complex carbohydrates, the host is also dependent on microbiota for maintaining blood pressure and vascular function

Hypertension is the single prominent risk factor of epidemic proportions leading to cardiovascular disease and stroke, which comprise the top two reasons for mortality of humans in the modern age. Much of the attention for the unknown causes of hypertension was focused on genetics and dietary salt, but in recent years, host-microbiotal interaction is gaining importance. Host-microbiotal partnership is key for the generation of many bioactive molecules including bile acid (BA) metabolites. Primary bile acids are synthesized and conjugated by the host but deconjugated and further modified to secondary BA by gut commensal bacteria. BA metabolites serve as important ligands for host nuclear receptors and/or G-protein-coupled receptors. These receptors have pivotal roles in blood pressure regulation. However, the effect of the host-microorganism biliary network on blood pressure (BP) remains poorly characterized. Here we report that both dietary salt and genetic factors rewire the composition of bile acids and BP. Specific reductions in conjugated bile acids were noted in human hypertensives as well as in rats with hypertension. Conjugation of bile acids by the host alone, devoid of the deconjugation step by microbiota, was sufficient to decrease BP of germ-free rats compared to germ-free conventionalized rats. Nutritional restoration of the conjugation of bile acids with Taurine increased the availability of circulating conjugated bile acids as ligands and ameliorated host susceptibility to hypertension via BA nuclear receptors and G-protein-coupled receptors. Thus, hosts and their bacterial symbionts can control host BP homeostasis via the resulting pool of bile acid metabolites. Sources of funding: National Institutes of Health (R01HL143082).

Mitochondria evolved from bacteria and use N-formylated peptides (NFPs) to synthetize protein. Bacterial and mitochondrial NFPs activate formyl peptide receptor 1 (FPR-1) and lead to vascular injury. We previously observed that Dahl Salt Sensitive rats (S) fed a low-salt (LS, 0.3% NaCl) diet presented spontaneous hypertension, vascular dysfunction, and overexpression of FPR-1 in arteries when compared to Dahl Salt Resistant (R) rats. High salt (HS, 2% NaCl) diet worsened these phenotypes in S rats. Interestingly, HS diet induced leaky gut and amoxicillin (AMO) treatment decreased BP in S-HS. Due to the dual sources of NFPs (microbiota and host mitochondria), we hypothesized that cell death-derived mitochondria and/or leaky gut-derived bacterial NFPs lead to FPR-1 activation, vascular injury and elevated BP in S rats independent of HS diet. For this, we used flow cytometry to measure cell necrosis and early and late apoptosis in kidney, bone marrow-derived macrophages and mesenteric resistance arteries (MRA) from male S and R rats (8-week old) on a LS diet. Zonulin, a biomarker for leaky gut, was measured in plasma. In another group, rats were treated with FPR-1 antagonist [Cyclosporin H (CsH), 0.3 mg/kg/day, osmotic mini-pump, 14 days], vehicle (VEH) or received water with AMO (5 mg/kg/day) for 21 days to deplete bacteria. BP was measured by telemetry and vascular function and structure were assessed in MRA. S rats presented increased kidney cell necrosis (R: 3.8±0.3 vs. S: 5.3±0.5* %). CsH decreased spontaneous elevation of BP [Diastolic: R+VEH: 77±2.7 vs. R+CsH: 81±1.2 vs. S+VEH: 126±3.0* vs. S+CsH:115±2.7 # ] and vascular hypercontractility [KCl (120mM): R+VEH: 9.4±1 vs. R+CsH: 10.2±0.4; S+VEH: 15.5±0.9* vs. S+CsH:11.7±0.8 # mN; Phenylephrine (10μM): R+VEH: 9.3±1 vs. R+CsH: 9.7±1; S+VEH: 14.5±1*vs. S+CsH: 11.4±0.6 # mN) in S-LS rats. AMO did not change vascular contraction or BP. Leaky gut was not observed in Dahl S-LS diet. In conclusion, FPR-1 can serve as a causative agent for the spontaneous elevation of BP and kidney-derived mitochondria, but not gut-derived microbiota, are the main source for NFPs.

Background: Recent evidence supports that metabolic dysfunction underlies hypertension. Injection of kynurenate, a metabolite of tryptophan pathway, into the paraventricular nucleus of the hypothalamus (PVN) lowers blood pressure (BP). Intestinal absorption and metabolism of tryptophan are impacted by gut microbiota. Since gut-brain axis contributes to gut dysbiosis-inducd hypertension, we hypothesized that gut microbiota modulates the levels of kynurenic pathway metabolites that have central impact on BP regulation. Methods: We, for the first time, used 7 weeks old male Germ-free (GF) Spague Dawley (SD) rats (n=5) and GF rats co-housed with conventional SD rats for 10 days (GFC) (n=6). BP was measured by tail-cuff. Serum metabolites were quantified by 6495 triple quandrople mass spectrometryand data was normalized using isotoplic labelled compounds. The nucleus of the solitary tract (NTS), the principal sensory nucleus for peripheral changes, and the PVN, a relay center projecting sympathetic output based on the integrated afferent inputs from brain regions including NTS, were analyzed by microarray hybridization for mRNA expression. Results: Compared to the GF rats, GFC rats had significantly higher systolic (139 mmHg vs 115 mmHg, p p p p Cd74, Il1b, Cxcl1, Mmp14 ) in the PVN (gene ontology analysis, p Sox11, Tp53, Cdk6, Hoxb4, Foxo4, Cyr61 ) in the NTS (gene ontology analysis, p Conclusion: Colonization of gut microbiota in GF rats induced increased cell differentiation and synaptic plasticity in the NTS and immune responses in the PVN, indicating the restructured sensory neurons of the NTS and enhanced sympathetic output from the PVN. These are in line with increased levels of kynurenic acid and 3-hydroxy kynurenine, and BP, respectively, suggesting that BP regulation by the gut-brain axis may be mediated by kynurenic pathway.

Aberrant immune responses are linked to hypertension; yet, neutrophils remain relatively understudied. Both neutrophilia and elevated neutrophil-to-lymphocyte (N/L) ratio are used as clinical biomarkers of hypertension. Likewise, neutrophilia is reported in spontaneously hypertensive rats. Whether these features are associated with an increase in neutrophil extracellular traps (NETs) generation is unknown. We hypothesized that neutrophilia and NETs directly correlate with hypertension whereby lowering neutrophilia and NETs are therapeutically beneficial to lower blood pressure (BP). Herein, we used various, genetic rat models divergent in BP to study the neutrophil-hypertension axis. These include the Dahl Salt-Sensitive (S) and Resistant (R) rats, and the low (LCR) and high (HCR) exercise capacity runner rats. LCR (2.1±0.3 10 9 /l) and S rats (1.2±0.5 10 9 /l) exhibited neutrophilia compared to HCR (0.98±0.5 10 9 /l) and R rats (1.04±0.5 10 9 /l), p

Background: Epoxides of arachidonic acid (EpETrEs, also commonly EETs) are potent anti-inflammatory lipid mediators and are associated with hypotensive and protective vascular actions. Marine omega-3 versions (epoxides of eicosapentaenoic and docosahexaenoic acids) have similar action. Surprisingly, none of these epoxides are as abundant in tissues or in plasma as epoxides of linoleic acid (EpOMEs) are. EpOMEs are up to one-hundred times more abundant than EpETrEs. Hence, EpOMEs could exert physiologic effects even with potency two-orders of magnitude higher, provided they have comparable efficacy. Few studies consider EpOMEs when assessing outcomes. Objective: To compare the potency and efficacy of EpOMEs to EpETrEs in a model of inflammation relevant to vascular disease, the RAW 264.7 macrophage. Approach and results: The suppression of inflammatory cytokines (IL-6 and TNFa) in RAW 264.7 macrophages responding to LPS treatment (500 nM, 24 hrs.) was measured in the presence of 1 hour pretreatment with 9(10)-EpOME and 11(12)-EpETrE over 4-orders of magnitude (0.03 nM to 100 nM). The resulting dose-response curves were used to estimate potency (IC 50 ), and efficacy (suppression as percent of maximal LPS activation). Least-square-mean and 95% CIs are reported. The IC 50 for suppression of cytokines by EpOME and EpETrE were not different, regardless of cytokine, yielding an estimate of 4.2 [2.6, 6.8] nM and indicating that EpOMEs are equipotent to EpETrEs in cytokine suppression. While EpOMEs had the same effectiveness in suppressing IL-6 to 59% [54, 64] of max, they were not as effective in suppressing TNFa, to 83% [80, 86] of max versus 78% [75, 81] for EpETrEs (p Conclusions: Given their great abundance in tissue and plasma, linoleate epoxides would need to be more than 100 times less potent than arachidonate epoxides to be physiologically irrelevant. Here, we find they are equipotent for suppression of two pro-inflammatory cytokines, with moderately lower efficacy for one cytokine, TNFa. We find little reason to ignore linoleate-derived EpOMEs and conclude they are a physiologically relevant pool of epoxides whose levels should be measured along with EpETrE and other epoxides when estimating overall epoxide effects.