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Background: Salt sensitivity of blood pressure is an independent predictor of cardiovascular morbidity and mortality. The exact mechanism by which salt intake increases blood pressure and cardiovascular risk is unknown. We previously found that sodium entry into antigen-presenting cells (APCs) via the amiloride-sensitive epithelial sodium channel EnaC (epithelial sodium channel) leads to the formation of IsoLGs (isolevuglandins) and release of proinflammatory cytokines to activate T cells and modulate salt-sensitive hypertension. In the current study, we hypothesized that ENaC-dependent entry of sodium into APCs activates the NLRP3 (NOD [nucleotide-binding and oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome via IsoLG formation leading to salt-sensitive hypertension. Methods: We performed RNA sequencing on human monocytes treated with elevated sodium in vitro and Cellular Indexing of Transcriptomes and Epitopes by Sequencing analysis of peripheral blood mononuclear cells from participants rigorously phenotyped for salt sensitivity of blood pressure using an established inpatient protocol. To determine mechanisms, we analyzed inflammasome activation in mouse models of deoxycorticosterone acetate salt–induced hypertension as well as salt-sensitive mice with ENaC inhibition or expression, IsoLG scavenging, and adoptive transfer of wild-type dendritic cells into NLRP3 deficient mice. Results: We found that high levels of salt exposure upregulates the NLRP3 inflammasome, pyroptotic and apoptotic caspases, and IL (interleukin)-1β transcription in human monocytes. Cellular Indexing of Transcriptomes and Epitopes by Sequencing revealed that components of the NLRP3 inflammasome and activation marker IL-1β dynamically vary with changes in salt loading/depletion. Mechanistically, we found that sodium-induced activation of the NLRP3 inflammasome is ENaC and IsoLG dependent. NLRP3 deficient mice develop a blunted hypertensive response to elevated sodium, and this is restored by the adoptive transfer of NLRP3 replete APCs. Conclusions: These findings reveal a mechanistic link between ENaC, inflammation, and salt-sensitive hypertension involving NLRP3 inflammasome activation in APCs. APC activation via the NLRP3 inflammasome can serve as a potential diagnostic biomarker for salt sensitivity of blood pressure.

Background: High salt consumption is associated with increased cardiovascular risk and higher morbidity and mortality in salt-sensitive hypertensives than in salt-resistant normotensives. Salt sensitivity of blood pressure (SSBP) is an independent predictor of death due to cardiovascular disease. Although the role of SMAD3 has been extensively studied in kidney fibrosis during renal artery stenosis and other cardiovascular disorders, the role of this pathway in immune cells contributing to SSBP is yet to be defined. Hypothesis: We hypothesized that antigen-presenting specific SMAD3, downstream of JAK2, mediates IsoLG-protein adducts formation, T cell activation, and inflammation and contributes to SSBP. Method. We enrolled two cohorts of participants. We isolated monocytes from cohort one, treated them with normal or high salt, and performed RNA-seq analysis. We used an inpatient salt load and salt depletion protocol to phenotype for salt-sensitive and salt-resistant participants in cohort 2 and performed CITE-Seq analysis. In additional experiments, we generated dendritic cell (DC)-specific JAK2 knockout mice (DCJAK2KO) and performed molecularly and flow cytometric immune phenotyping along with both noninvasive tail-cuff and state-of-the-art radio telemetry blood pressure and heart rate (HR) monitoring in the L-NAME/high salt model of salt sensitivity. We used immunohistochemistry and Fluorescent In Situ Hybridization for spatial and differential infiltration of DCs, and expression of JAK2, SMAD3, ENAC-γ, and IL-6 in DCs as well as fibrosis and macrophages in the kidney. Results: Both bulk and single-cell transcriptomic analyses of human myeloid antigen-presenting cells revealed that high salt treatment in vitro and in vivo upregulates genes of the JAK-STAT-SMAD pathway and downregulates downstream regulators, including the suppressor of cytokine signaling (SOCS) genes. DCJAK2KO mice exhibited attenuated salt-sensitive hypertension ( Systolic blood pressure, 121.6 vs. 138.5, SEM±3.3, n=6) and reduced HR compared to the wildtype littermates during L-NAME/high salt regimen. This was associated with reduced phosphorylation/activation of SMAD3 in total leukocytes ( 982.6 vs 434.7, SEM±107.3), DCs (63.6 vs 18.8, SEM±10.73), and monocytes ( 17.5 vs 106.2, SEM±34.1). Inflammatory markers, IsoLG-protein adducts ( 7.1 vs 25.9, SEM±3.6), IL-6 (6.8 vs 26, SEM±5.9), and TGF-β1 ( 38 vs 91.8, SEM±26.4) in DCs were significantly attenuated. Similarly, these markers were downregulated in total leukocytes and monocytes. The CD8a+ central memory T (TCM) and effector memory T (TEM) cells exhibited lowered IL-17A ( 14.6 vs 26.3, SEM±5.1; 5.7 vs 38.5, SEM±13.96) and IFN-γ ( 26.3 vs 1.104.7, SEM±59.01; 11.6 vs 52.3, SEM±15.6) expression. DCJAK2KO mice also showed attenuated infiltration of total leukocytes, DCs, monocytes, and lymphocytes in the kidney. Conclusion: These results indicate that DC-specific SMAD3 downstream of JAK2 plays an essential role in SSBP. 1K01HL130497-01 (Kirabo, PI); 1R01HL144941-01A1 (Kirabo, PI); 1R03HL155041-01 (Kirabo, PI); 5R01HL147818-22 (Kleyman & Kirabo, MPI); AHA 903428 (Ishimwe). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Salt-sensitivity of blood pressure puts both normotensive and hypertensive individuals at a higher risk for cardiovascular morbidity and mortality. Isolevuglandins (IsoLGs) have been shown to be increased in multiple oxidative-stress associated diseases, including salt-sensitive hypertension. We had previously reported that mice who received adoptive transfer of DCs exposed to high salt exhibited a prohypertensive response, which was abolished when these DCs were pretreated with IsoLG specific scavenger, 2-HOBA. Additionally, we found that scavenging of IsoLGs using 2-HOBA in a mouse model of salt-sensitive hypertension blocked NLRP3 inflammasome in dendritic cells (DCs). Whether IsoLGs directly lead to NLRP3 inflammasome activation remain unknown. IsoLGs have been shown to adduct to histones, specifically H4 histones. Histones are responsible for regulating chromatin structure. However, it is unknown how IsoLG-modified histones affect gene transcription. We tested the hypothesis that elevated sodium in APCs leads to IsoLG-adducted H4 histones increasing transcriptional access to NLRP3 inflammasome components contributing to salt-sensitive hypertension. Methods: In vitro studies were performed on isolated monocytes from 11 hypertensive individuals treated with either normal salt (150mM) or high salt (190mM) and submitted for RNA sequencing. In vivo studies were performed on 19 hypertensive subject enrolled in an acute inpatient protocol to assess response to salt loading (460 mmoL/24 hours) and salt depletion (10 mmoL/24 hours, and furosemide 40 mg x 3). IsoLG-containing DCs were identified using flow cytometry as CD45+MerTK-HLADR+CD1c+. Isolated PBMCs from a salt sensitive subject (ΔSBPSD-SL = -12.9) and a salt resistant subject (ΔSBPSD-SL = 0.35) were analyzed using assay for transposase-accessible chromatin with high-throughput sequencing (ATACseq). Results: Using RNA sequencing data of isolated monocytes and the EpiFactors database, I identified differentially expressed genes related to histones and histone modification. This analysis demonstrated an overall upregulation in histone modification after high salt visualized in a heatmap and PCA plot, specifically upregulation of gene HIST4H4 which encodes the Histone H4 (25.4 ± 4.6 vs 65.5 ± 6.5, [n=11, q=1.5 x 10-3, paired analyses and adjusted P values (q), false discovery rate (+] increase in IsoLG-adducts may epigenetically regulate NLRP3 inflammasome components in SSBP. No Disclosures. Funding Sources: K01HL130497, R01HL147818, R03HL155041, R01HL144941 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PURPOSE OF REVIEW: Kidney disease is a strong modulator of the composition and metabolism of the intestinal microbiome that produces toxins and inflammatory factors. The primary pathways for these harmful factors are blood vessels and nerves. Although lymphatic vessels are responsible for clearance of interstitial fluids, macromolecules, and cells, little is known about whether and how kidney injury impacts the intestinal lymphatic network. RECENT FINDINGS: Kidney injury stimulates intestinal lymphangiogenesis, activates lymphatic endothelial cells, and increases mesenteric lymph flow. The mesenteric lymph of kidney-injured animals contains increased levels of cytokines, immune cells, isolevuglandin (IsoLG), a highly reactive dicarbonyl, and of apolipoprotein AI (apoAI). IsoLG is increased in the ileum of kidney injured animals, and intestinal epithelial cells exposed to myeloperoxidase produce more IsoLG. IsoLG-modified apoAI directly increases lymphatic vessel contractions and activates lymphatic endothelial cells. Inhibition of IsoLG by carbonyl scavenger treatment reduces intestinal lymphangiogenesis in kidney-injured animals. Research from our group and others suggests a novel mediator (IsoLG-modified apoAI) and a new pathway (intestinal lymphatic network) in the cross talk between kidneys and intestines and heart. SUMMARY: Kidney injury activates intestinal lymphangiogenesis and increases lymphatic flow via mechanisms involving intestinally generated IsoLG. The data identify a new pathway in the kidney gut–heart axis and present a new target for kidney disease-induced intestinal disruptions that may lessen the major adverse consequence of kidney impairment, namely cardiovascular disease.

Significance: Salt sensitivity of blood pressure (SSBP) is an independent risk factor for mortality and morbidity due to cardiovascular disease, and disproportionately affects blacks and women. Several mechanisms have been proposed, including exaggerated activation of sodium transporters in the kidney leading to salt retention and water. Recent Advances: Recent studies have found that in addition to the renal epithelium, myeloid immune cells can sense sodium via the epithelial Na(+) channel (ENaC), which leads to activation of the nicotinamide adenine dinucleotide phosphate oxidase enzyme complex, increased fatty acid oxidation, and production of isolevuglandins (IsoLGs). IsoLGs are immunogenic and contribute to salt-induced hypertension. In addition, aldosterone-mediated activation of ENaC has been attributed to the increased SSBP in women. The goal of this review is to highlight mechanisms contributing to SSBP in blacks and women, including, but not limited to increased activation of ENaC, fatty acid oxidation, and inflammation. Critical Issues: A critical barrier to progress in management of SSBP is that its diagnosis is not feasible in the clinic and is limited to expensive and laborious research protocols, which makes it difficult to investigate. Yet without understanding the underlying mechanisms, this important risk factor remains without treatment. Future Directions: Further studies are needed to understand the mechanisms that contribute to differential blood pressure responses to dietary salt and find feasible diagnostic tools. This is extremely important and may go a long way in mitigating the racial and sex disparities in cardiovascular outcomes. Antioxid. Redox Signal. 35, 1477–1493.

Salt sensitivity of blood pressure is an independent cardiovascular risk factor as powerful as hypertension, yet it goes untreated due to the lack of a feasible diagnostic tool. We previously found that sodium enters antigen-presenting cells (APCs) via ENaC, leading to the formation of isolevuglandins (IsoLGs). IsoLGs are highly reactive oxidative products of fatty acids and act as neoantigens to trigger inflammation in hypertension. We found that activation of APCs via IsoLG is highly variable in humans. Thus, we hypothesized that this variability in IsoLG-mediated APC activation contributes to the variability observed in human salt sensitivity of blood pressure and that IsoLGs can be used as a diagnostic tool. We measured systolic blood pressure (SBP) and IsoLG-containing APCs (dendritic cells [DC], classical, intermediate and non-classical monocytes) by flow cytometry in 14 hypertensive subjects who were off therapy for 2 weeks, before (B) and after in-patient 24 hr salt loading (HI, 460 mmol Na + ) and salt depletion (LO, 10 mmol Na + and furosemide 40 mg x 3). Salt sensitivity was assessed by ΔSBP (LO minus HI). Median age was 49, with 5 females, median BMI (IQR) of 29.8 (25.2, 39.5) kg/m 2 and screening SBP of 135.1 (130, 144.9) mmHg. The median urinary Na + excretions (IQR) were 169.6 (125, 203.3), 360.9 (324.1, 421.2) and 54.8 (36.8, 76) mmol/24h at baseline, HI and LO days, respectively. Baseline percentages of classical and non-classical monocytes with IsoLGs were positively correlated with ΔSBP (r=0.64, p=0.01 and r=0.71, p

Background: African Americans (AAs) are disproportionately affected by cardiovascular disease (CVD), they are 20% more likely to die from CVD than whites, chronic exposure to inflammation and oxidative stress contributes to CVD. In previous studies, enhancing parasympathetic cholinergic activity has been shown to decrease inflammation. Considering that AAs have decreased parasympathetic activity compared to whites, we hypothesize that stimulating it with a central acetylcholinesterase (AChE) inhibitor, galantamine, would prevent lipid-induced oxidative stress.Objective: To test the hypothesis that acute dose of galantamine, an AChE inhibitor, decreases lipid-induced oxidative stress in obese AAs.Methods: Proof-of-concept, double-blind, randomized, placebo-controlled, crossover study that tested the effect of a single dose of 16 mg of galantamine versus placebo on lipid-induced oxidative stress in obese AAs. Subjects were studied on two separate days, one week apart. In each study day, 16 mg or matching placebo was administered before 20% intralipids infusion at doses of 0.8 mL/m2/min with heparin at doses of 200 U/h for 4 hours. Outcomes were assessed at baseline, 2 and 4 hours during the infusion.Main Outcome Measures: Changes in F2-isoprostane (F2-IsoPs), marker of oxidative stress, measured in peripheral blood mononuclear cells (PBMC) and in plasma at baseline, 2, and 4-hrs post-lipid infusion. Secondary outcomes include changes in inflammatory cytokines (IL-6, TNF alpha).Results: A total of 32 obese AA women were screened and fourteen completed the study (age 37.8±10.70 years old, BMI 38.7±3.40 kg/m2). Compared to placebo, 16 mg of galantamine significantly inhibited the increase in F2-IsoPs in PBMC (0.007±0.008 vs. -0.002±0.006 ng/sample, P=0.016), and plasma (0.01±0.02 vs. -0.003±0.01 ng/mL, P=0.023). Galantamine also decreased IL-6 (11.4±18.45 vs. 7.7±15.10 pg/mL, P=0.021) and TNFα levels (18.6±16.33 vs. 12.9±6.16 pg/mL, P=0.021, 4-hrs post lipid infusion) compared with placebo. These changes were associated with an increased plasma acetylcholine levels induced by galantamine (50.5±10.49 vs. 43.6±13.38 during placebo pg/uL, P=0.025).Conclusions: In this pilot, proof-of-concept study, enhancing parasympathetic nervous system (PNS) cholinergic activity with galantamine inhibited lipid-induced oxidative stress and inflammation induced by lipid infusion in obese AAs.Trial registration: ClinicalTrials.gov identifiers NCT02365285

The epithelial Na(+) channel (ENaC) promotes the absorption of Na(+) in the aldosterone-sensitive distal nephron, colon, and respiratory epithelia. Deletion of genes encoding subunits of ENaC results in early postnatal mortality. Here, we present the initial characterization of a mouse with dramatically suppressed expression of the ENaC γ-subunit. We used this hypomorphic (γ(mt)) allele to explore the importance of this subunit in homeostasis of electrolytes and body fluid volume. At baseline, γ-subunit expression in γ(mt/mt) mice was markedly suppressed in the kidney and lung, whereas electrolytes resembled those of littermate controls. Aldosterone levels in γ(mt/mt) mice exceeded those seen in littermate controls. Quantitative magnetic resonance measurement of body composition revealed similar baseline body water, lean tissue mass, and fat tissue mass in γ(mt/mt) mice and controls. γ(mt/mt) mice exhibited a more rapid decline in body water and lean tissue mass in response to a low-Na(+) diet than the controls. Replacement of drinking water with 2% saline selectively and transiently increased body water and lean tissue mass in γ(mt/mt) mice relative to the controls. Lower blood pressures were variably observed in γ(mt/mt) mice on a high-salt diet compared with the controls. γ(mt/mt) also exhibited reduced diurnal blood pressure variation, a “nondipping” phenotype, on a high-Na(+) diet. Although ENaC in the renal tubules and colon works to prevent extracellular fluid volume depletion, our observations suggest that ENaC in other tissues may participate in regulating extracellular fluid volume and blood pressure. NEW & NOTEWORTHY A mouse with globally suppressed expression of the epithelial Na(+) channel γ-subunit showed enhanced sensitivity to dietary salt, including a transient increase in total body fluid, reduced blood pressure, and reduced diurnal blood pressure variation when given a dietary NaCl challenge. These results point to a role for the epithelial Na(+) channel in regulating body fluid and blood pressure beyond classical transepithelial Na(+) transport mechanisms.

Background: High Na+ stimulates antigen-presenting cells (APCs) in an ENaC dependent manner, with formation of isolevuglandin (isoLG) adducts (neoantigen peptides) that promote T cell activation and salt sensitive (SS) hypertension in rodents. Methods: We studied this pathway in 9 subjects with essential hypertension who discontinued anti-hypertensive therapy for 2 weeks. Their SS was assessed by 24-hrs of salt loading (460 mmoL) and salt depletion (10 mmoL/24 hr, plus furosemide 40 mg x 3). Muscle and skin Na + were measured at baseline (BA) by 23 Na magnetic resonance imaging (NaMRI). The % of APCs containing isoLG adducts (flow cytometry), urine and serum electrolytes and epoxyeicosatrienoic acids (EETs 8-9, 11-12 and 14-15) were measured at BA, after salt-loading (HI) and after salt-depletion (LO). Results: Age was 54 years (48-56), with 23% female, BMI 30 kg/m 2 (28-40) and screening SBP 136 mmHg (120-144), and DBP 85 mmHg (75-99). BA 24-hr urine Na + excretion was 178 (143-212) mmoL, Hi 392 (229-421) and LO 27 (25-29). SBP response to salt-depletion varied from -13.8 to +5.6 mmHg. Muscle Na+ correlated with duration of hypertension (r=0.73, p Conclusions: Muscle Na+ increases with duration of hypertension and correlates with severity of BP elevation. Changes in APC isoLGs due to Na+ loading or depletion seem to be a biomarker of SS of BP in humans. Relations between urine EETs and ΔEETs with APC isoLGs and ΔisoLGs suggest that EETs might be inhibitors of APC ENaC as they are of renal ENaC. Relationships between isoLGs and urine but not plasma EETs suggest that activation of APCs by high salt may occur in the hyperosmolar renal medulla.

Salt sensitivity of blood pressure is an independent predictor of death due to cardiovascular events. Diagnosis of salt-sensitivity is not feasible in the clinic, making it difficult to investigate therapeutic strategies. We hypothesized that NLRP3-inflammasome and IL-1β production in monocytes plays a role in salt-sensitive hypertension. We phenotyped salt-sensitivity of blood pressure using an acute inpatient Weinberger protocol of an isocaloric high salt diet and rapid intravenous salt-loading, followed by low salt diet and furosemide-induced salt-depletion. Ambulatory blood pressure was continuously monitored and averaged for the days of salt-loading and salt-depletion. Blood samples were obtained at baseline, salt-loading, and after salt-depletion. Median age was 54 years (44-55), 3 of the 5 subjects were female, screening systolic blood pressure was 140 mmHg (134-148), diastolic blood pressure was 88 mmHg (84-99), and BMI was 35 kg/m 2 (30-39). Using cell hashing and ECCITE-seq analysis, we profiled transcriptomes in multiple immune cell types using antibody-derived tags (ADTs). UMAP clustering of different cell types were identified by ADTs including monocyte markers CD14 and CD16. Interestingly, UMAP visualization of CD14+ and CD16+ clusters indicated a greater decrease in CD14+ clusters after salt-depletion in the salt-resistant subjects than the salt-sensitive; however the salt-sensitive subjects had a greater decrease in CD16+ clusters than the salt-resistant group after both salt-loading and salt-depletion. These data were confirmed using flow cytometry. Unlike in salt-resistant participants, we found that within monocyte clusters, salt-sensitivity was associated with down regulation of the inflammasome components NLRP3 (0.386 ± 1.18 vs. 0.197 ± 0.778) and IL-1β (0.858 ± 2.32 vs. 0.159 ± 0.925) following salt-depletion. Using flow cytometry, we found Δ% isoLG+ CD14+/CD16+ monocytes correlated with salt-sensitivity of blood pressure (r=0.88, 95% CI, p=0.05). These results suggest that the inflammasome and monocyte activation are dynamically regulated by dietary salt in vivo and can serve as a potential diagnostic biomarker for salt-sensitivity of blood pressure.

Elevated cardiovascular risk including stroke, heart failure, and heart attack is present even after normalization of blood pressure in patients with hypertension. Underlying immune cell activation is a likely culprit. Although immune cells are important for protection against invading pathogens, their chronic overactivation may lead to tissue damage and high blood pressure. Triggers that may initiate immune activation include viral infections, autoimmunity, and lifestyle factors such as excess dietary salt. These conditions activate the immune system either directly or through their impact on the gut microbiome, which ultimately produces chronic inflammation and hypertension. T cells are central to the immune responses contributing to hypertension. They are activated in part by binding specific antigens that are presented in major histocompatibility complex molecules on professional antigen-presenting cells, and they generate repertoires of rearranged T-cell receptors. Activated T cells infiltrate tissues and produce cytokines including interleukin 17A, which promote renal and vascular dysfunction and end-organ damage leading to hypertension. In this comprehensive review, we highlight environmental, genetic, and microbial associated mechanisms contributing to both innate and adaptive immune cell activation leading to hypertension. Targeting the underlying chronic immune cell activation in hypertension has the potential to mitigate the excess cardiovascular risk associated with this common and deadly disease.

Introduction: Inflammation and oxidative stress are linked to multiple risk factors for atrial fibrillation (AF), and to AF itself in the setting of sterile injury (e.g. after catheter ablation or cardiac surgery). However, anti-inflammatory therapies and conventional antioxidants cause adverse effects or are ineffective to prevent AF. Highly reactive mediators of lipid peroxidation such as isolevuglandins (IsoLGs) have been identified as a major component of oxidative stress-related injury. We hypothesized that during AF promoted by cardiac inflammation, a scavenger of IsoLG will decrease AF susceptibility. Methods: We studied mice with a systemic inflammatory phenotype due to deficiency in the lymphocyte adaptor protein ( Lnk -/- ), a negative regulator of cytokine signaling. At weaning, Lnk -/- mice and their wild-type (WT) littermates received either vehicle or a potent IsoLG scavenger, 2-hydroxybenzylamine (2-HOBA), by oral administration. At age 14 weeks, animals underwent transesophageal burst pacing, echocardiography, and tissue harvest or flow cytometry to measure atrial inflammation and IsoLG-adducts. Results: Cardiac histology and echocardiography revealed no major histologic or structural abnormalities in Lnk -/- mice. Nevertheless, Lnk -/- mice demonstrated a significant increase in AF burden compared to WT controls (124.8±43.3 vs 6.8±3 sec, respectively [mean±SEM, n=28, 12; P 30 sec; 48.1% vs 0%; P-/- mice, with a significant increase in CD3, CD19, NK1.1, and CD11b/MHCII positive cells, compared to atria from WT control mice. Furthermore, there was a 2 to 4-fold increase in IsoLG-adducts for Lnk -/- atrial immune cells positive for CD3, CD19, NK1.1 and CD11b/MHCII, compared to cells from WT atria. Lnk -/- mice treated with 2-HOBA had significantly reduced AF burden (4.7±4.5 sec, n=7; P Conclusions: IsoLGs play a critical role in the pathogenesis of inflammation-mediated AF, and 2-HOBA, a scavenger of IsoLGs, represents a potentially novel therapeutic strategy for AF in this clinical setting.

Purpose of Review Salt sensitivity of blood pressure (SSBP) is an independent predictor of death due to cardiovascular events and affects nearly 50% of the hypertensive and 25% of the normotensive population. Strong evidence indicates that reducing sodium (Na+) intake decreases blood pressure (BP) and cardiovascular events. The precise mechanisms of how dietary Na+ contributes to elevation and cardiovascular disease remain unclear. The goal of this review is to discuss mechanisms of salt-induced cardiovascular disease and how the microbiome may play a role. Recent Findings The innate and adaptive immune systems are involved in the genesis of salt-induced hypertension. Mice fed a high-salt diet exhibit increased inflammation with a marked increase in dendritic cell (DC) production of interleukin (IL)-6 and formation of isolevuglandins (IsoLG)-protein adducts, which drive interferon-gamma (IFN-γ) and IL-17A production by T cells. While prior studies have mainly focused on the brain, kidney, and vasculature as playing a role in salt-induced hypertension, the gut is the first and largest location for Na+ absorption. Research from our group and others strongly suggests that the gut microbiome contributes to salt-induced inflammation and hypertension. Summary Recent studies suggest that alterations in the gut microbiome contribute to salt-induced hypertension. However, the contribution of the microbiome to SSBP and its underlying mechanisms are not known. Targeting the microbiota and the associated immune cell activation could conceivably provide the much-needed therapy for SSBP.

Introduction: Salt Sensitivity (SS) of blood pressure (BP) is an independent predictor of death due to cardiovascular disease, but its pathogenesis is poorly understood. Sodium (Na + ) is stored in the skin and muscle interstitium. This hyperosmolar Na + activates monocytes in vitro via oxidative stress with generation of isolevuglandin (isoLG) protein adducts that are immunogenic and activate the adaptive immune system. Methods: Five subjects with essential hypertension discontinued all anti-hypertensive therapy for two weeks before the study. SS was assessed by an inpatient protocol of salt loading (460 mmoL/24h) and salt depletion (10 mmoL/24h, plus furosemide 40 mg x 3). Muscle and skin Na + contents were measured at baseline (BA) by 23 Sodium magnetic resonance imaging ( 23 NaMRI). Urine and serum electrolytes, glomerular filtration rate and the % CD14 + monocytes containing isoLG adducts using flow cytometry were obtained at BA, after salt-loading (HI) and after salt-depletion (LO). All continuous data are displayed as median (interquartile range). Spearman’s correlation was used to test associations. Results: Median age was 54 years (44-55), 60% of subjects were female, screening systolic BP (SBP) was 140 mmHg (134-148), diastolic BP was 88 mmHg (84-99) and BMI was 35 kg/m 2 (30-39). SBP response to salt-depletion (salt-sensitivity index, SSI) varied from -13.8 to +1.8 mmHg. %isoLG + CD14 + cells were 48 (27-65) at BA, 55 (31-56) at HI, and 70 (33-72) at LO (p=0.594, by the Kruskal-Wallis test). The correlation between SSI and delta (Δ) %isoLG LO minus HI, was 0.86, [95% confidence interval (CI), -0.07-0.99] which may suggest conclusively as we gather more data that the greater the SSI, the larger the decrease in isoLGs by salt depletion. Muscle Na + content correlated with 24h urine Na + (BA) (r=0.90, 95% CI, 0.11-0.99), however, the correlation with BP, SSI or isoLGs was inconclusive, potentially due to the small sample size. Skin Na + content correlated with baseline %CD14IsoLG + (r=0.91; 95% CI, 0.12-0.99). Conclusions: Na + intake is a component of the determinants of muscle Na + . Skin Na + is associated with increased isoLGs in monocytes, a marker of immune cell activation. Variability in ΔCD14isoLG may serve as a biomarker for SS of BP in humans.

Salt-sensitivity is a major risk factor for hypertension and cardiovascular disease (CVD). Reducing dietary Na + decreases blood pressure and CVD risk. However, the precise mechanisms of how Na + leads to hypertension are still not well defined. Recently, we found that dendritic cells (DCs) in response to increases in extracellular [Na + ] exhibit an amiloride-sensitive epithelial Na + channel (ENaC)-dependent activation of NADPH-oxidase, superoxide production, reactive isolevuglandin (IsoLG)-protein adduct formation, and cytokine secretion which promote hypertension. We hypothesized that the NLRP3 inflammasome in antigen-presenting cells (APCs) mediates salt-sensitive hypertension through an ENaC-dependent mechanism. To test this hypothesis, we cultured mouse splenocytes in normal-salt or high-salt (HS) media with or without co-treatment with the ENaC inhibitor, amiloride (20 μM). Using flow cytometry, we found that HS increased monocyte and DC IL-1β production, which was confirmed through an ELISA assay detecting IL-1β release (2.131 ± 0.733 vs 12.75 ± 1.108 pg/mL, pin vitro data, we treated salt-sensitive mice on a 129-SvJ background with a HS diet (4% NaCl) for 28 days with or without amiloride (1mg/kg/day in drinking water) or NLRP3 inflammasome inhibitor MCC950 (10mg/kg i.p.). Amiloride or MCC950 treated mice developed blunted hypertension in response to HS (120.4 ± 2.99; 101.0 ± 3.74) compared to vehicle controls (140.5 ± 3.98). Amiloride treated mice also exhibited less expression of NLRP3, pro-IL1β, and IsoLGs in DCs and monocytes compared to controls. Interestingly, MCC950 treated mice exhibited decreased pro-IL1β but not NLRP3 expression or IsoLG production. Using the DOCA-salt model, we found similar increases in NLRP3, pro-IL1β, and IsoLGs expression in DCs and monocytes, which was abolished after treatment with IsoLG scavenger 2-HOBA (1g/L). Our findings suggest a role for ENaC-dependent NLRP3 inflammasome activation in APCs in response to a HS diet, which may represent a promising treatment approach to salt-induced hypertension.

Hypertension is a major risk factor for development of cardiovascular disease. Excess dietary salt contributes to inflammation and the genesis of hypertension. We recently found that gamma and alpha subunits of the epithelial sodium channel (ENaCαγ) on dendritic cells mediate NADPH oxidase-dependent formation of immunogenic isolevuglandin (IsoLG)-protein adducts leading to inflammation and salt-sensitive hypertension. We hypothesized that Jak2 expression, specifically in CD11c + myeloid cells, regulates expression of ENaCγ and promotes salt-sensitive hypertension. Our results indicate that deletion of Jak2 in CD11c + myeloid cells reduced the salt-induced expression of ENaCγ in CD11c+ cells. Moreover, mice lacking Jak2 in CD11c+ cells developed a blunted hypertensive response (123.8±4.7) during the high salt feeding phase of the N-Nitro-L-arginine methyl ester hydrochloride (L- NAME)/high salt model of salt-sensitive hypertension compared to their wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention (69.55±5.8) in response to high salt-feeding when compared to the wildtype litter mate controls (57.89±9.5). These results indicate that Jak2 expression in CD11c + myeloid cells plays a role in salt- sensitive hypertension through an ENaC-dependent mechanism.

Purpose of Review The main goal of this article is to discuss the role of the epithelial sodium channel (ENaC) in extracellular fluid and blood pressure regulation. Recent Findings Besides its role in sodium handling in the kidney, recent studies have found that ENaC expressed in other cells including immune cells can influence blood pressure via extra-renal mechanisms. Dendritic cells (DCs) are activated and contribute to salt-sensitive hypertension in an ENaC-dependent manner. We discuss recent studies on how ENaC is regulated in both the kidney and other sites including the vascular smooth muscles, endothelial cells, and immune cells. We also discuss how this extra-renal ENaC can play a role in salt-sensitive hypertension and its promise as a novel therapeutic target. Summary The role of ENaC in blood pressure regulation in the kidney has been well studied. Recent human gene sequencing efforts have identified thousands of variants among the genes encoding ENaC, and research efforts to determine if these variants and their expression in extra-renal tissue play a role in hypertension will advance our understanding of the pathogenesis of ENaC-mediated cardiovascular disease and lead to novel therapeutic targets.

Aims Prior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro. Methods and results To study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments. Exposure of human monocytes to elevated Na+ex vivo caused a co-ordinated response involving isolevuglandin (IsoLG)-adduct formation, acquisition of a dendritic cell (DC)-like morphology, expression of activation markers CD83 and CD16, and increased production of pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-6, and IL-1β. High salt also caused a marked change in monocyte gene expression as detected by RNA sequencing and enhanced monocyte migration to the chemokine CC motif chemokine ligand 5. NADPH-oxidase inhibition attenuated monocyte activation and IsoLG-adduct formation. The increase in IsoLG-adducts correlated with risk factors including body mass index, pulse pressure. Monocytes exposed to high salt stimulated IL-17A production from autologous CD4+ and CD8+ T cells. In addition, to evaluate the effect of salt in vivo, monocytes and T cells isolated from humans were adoptively transferred to immunodeficient NSG mice. Salt feeding of humanized mice caused monocyte-dependent activation of human T cells reflected by proliferation and accumulation of T cells in the bone marrow. Moreover, we performed a cross-sectional study in 70 prehypertensive subjects. Blood was collected for flow cytometric analysis and 23Na magnetic resonance imaging was performed for tissue sodium measurements. Monocytes from humans with high skin Na+ exhibited increased IsoLG-adduct accumulation and CD83 expression. Conclusion Human monocytes exhibit co-ordinated increases in parameters of activation, conversion to a DC-like phenotype and ability to activate T cells upon both in vitro and in vivo sodium exposure. The ability of monocytes to be activated by sodium is related to in vivo cardiovascular disease risk factors. We therefore propose that in addition to the kidney and vasculature, immune cells like monocytes convey salt-induced cardiovascular risk in humans., Graphical Abstract

 Animal models for atherogenic changes in coronary arterial wall or CAMs were produced, and primary cell culture of CAMs, Western blot analysis, and confocal microscopy of autophagic and dedifferentiation markers were conducted as we described previously (J Cell Mol Med. 2014; 18: 2165-2175).  Cell cycle analysis and proliferation rate determination were performed as described in previous studies (Plos One 2015; 10: D119506)

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