No abstract

Salt-sensing mechanisms in hypertension involving the kidney, vasculature, and central nervous system have been well studied; however, recent studies suggest that immune cells can sense sodium (Na + ). Antigen-presenting cells (APCs) including dendritic cells critically modulate inflammation by activating T cells and producing cytokines. We recently found that Na + enters dendritic cells through amiloride-sensitive channels including the α and γ subunits of the epithelial sodium channel (ENaC) and mediates nicotinamide adenine dinucleotide phosphate oxidase-dependent formation of immunogenic IsoLG (isolevuglandin)-protein adducts leading to inflammation and hypertension. Here, we describe a novel pathway in which the salt-sensing kinase SGK1 (serum/glucocorticoid kinase 1) in APCs mediates salt-induced expression and assembly of ENaC-α and ENaC-γ and promotes salt-sensitive hypertension by activation of the nicotinamide adenine dinucleotide phosphate oxidase and formation of IsoLG-protein adducts. Mice lacking SGK1 in CD11c + cells were protected from renal inflammation, endothelial dysfunction, and developed blunted hypertension during the high salt feeding phase of the N-Nitro-L-arginine methyl ester hydrochloride/high salt model of salt-sensitive hypertension. CD11c + APCs treated with high salt exhibited increased expression of ENaC-γ which coimmunoprecipitated with ENaC-α. This was associated with increased activation and expression of various nicotinamide adenine dinucleotide phosphate oxidase subunits. Genetic deletion or pharmacological inhibition of SGK1 in CD11c + cells prevented the high salt-induced expression of ENaC and nicotinamide adenine dinucleotide phosphate oxidase. These studies indicate that expression of SGK1 in CD11c + APCs contributes to the pathogenesis of salt-sensitive hypertension.

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.

Inflammation has been implicated in the pathogenesis of hypertension and recent evidence suggests that isolevuglandin (IsoLG)-protein adducts play a role. Several hypertensive stimuli contribute to formation of IsoLG-protein adducts including excess dietary salt and catecholamines. The precise intracellular mechanisms by which these hypertensive stimuli lead to IsoLG-protein adduct formation are still not well understood; however, there is now evidence implicating NADPH-oxidase derived reactive oxygen species (ROS) in this process. ROS oxidize arachidonic acid leading to formation of IsoLGs, which non-covalently adduct to lysine residues and alter protein structure and function. Recent studies suggest that these altered proteins act as neo-antigens leading to an autoimmune state that results in hypertension. The goal of this mini-review is to highlight some of the hypertensive stimuli and the mechanisms contributing to IsoLG-protein adduct formation leading to inflammation and hypertension.

Considerable evidence demonstrates that excess dietary sodium (Na+) is a major independent risk factor for hypertension and CVD in both hypertensive and normotensive subjects. Approximately 50% of hypertensive patients are salt‐sensitive, and reducing dietary sodium (Na+) decreases both blood pressure and CVD risk. The precise mechanisms of how salt leads to hypertension are still not well defined. The amiloride‐sensitive epithelial Na+ channel (ENaC) in non‐epithelial cells have been found to play a role in blood pressure regulation. Recently, our lab has shown that dendritic cells (DCs) in response to increases in extracellular [Na+] exhibit an ENaC‐dependent activation of NADPH‐oxidase, superoxide production, reactive isolevuglandin (IsoLG)‐protein adduct formation, and cytokine secretion which promote hypertension. In this study, 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 media with or without co‐treatment with the ENaC inhibitor, amiloride (20 μM). Using flow cytometry, we found that high salt increased both monocyte and DC production of IL‐1β, which was confirmed through an ELISA assay detecting release of IL‐1β (2.131 ± 0.733 vs 12.75 ± 1.108 pg/mL, p<0.0001) into the culture media. Treatment with amiloride prevented production of IL‐1β (12.75 ± 1.108 vs 1.905 ± 0.3495 pg/mL, p<0.0001) in both monocytes and DCs. To confirm our in vitro data, we treated salt‐sensitive mice on a 129‐SvJ background with a high‐salt diet (4% NaCl) for 28 days with or without ENaC inhibitor amiloride (1mg/kg/day in drinking water) or NLRP3 inflammasome inhibitor MCC950 (10mg/kg i.p.). Mice treated with amiloride or MCC950 developed blunted hypertension (120.4 ± 2.99; 101.0 ± 3.74) compared to the high‐salt treated controls (140.5±3.98). Mice treated with amiloride also exhibited less expression of NLRP3, pro‐IL1b, and IsoLGs in both DCs and monocytes. Interestingly, the MCC950 treated mice only exhibited decreased pro‐IL1b but not NLRP3 expression or IsoLG production. Using the deoxycorticosterone acetate and 1% salt diet (DOCA‐salt) model, we found similar increases in expression of NLRP3, pro‐IL1b, and IsoLGs in DCs and monocytes as the 4% NaCl high salt diet fed mice. Treatment of the DOCA‐salt mice with the IsoLG scavenger 2‐HOBA (1g/L) attenuated expression of NLRP3, pro‐IL1b, and IsoLGs in both DCs and monocytes. Our findings suggest a role for ENaC‐dependent NLRP3 inflammasome activation in APCs in response to a high‐salt diet, which may represent a promising approach to treatment of salt‐induced hypertension. Support or Funding Information K01HL130497, R01HL147818, T32HL144446

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 h. Outcomes were assessed at baseline, 2 and 4 h 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-h 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-h 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.

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.

Salt‐sensitivity of blood pressure affects 50% of hypertensive and 25% of normotensive individuals and is an independent predictor of death due to cardiovascular disease. 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 expression of Jak2 specifically in antigen presenting myeloid cells contributes to salt‐sensitive hypertension in an ENaC dependent mechanism. Profiling whole transcriptome using RNA‐Seq and analyzing the gene differential expression patterns in human monocytes reveals that high salt treatment upregulates genes of the Jak/STAT pathway, and the downstream regulators suppressor of cytokine signaling (SOCS) genes. Male and female mice lacking Jak2 in CD11c+ cells developed blunted hypertension (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 the wildtype littermate controls (140.5±6.5). These mice also exhibited less infiltration of monocyte/macrophages in their kidneys and less volume retention in response to high salt‐feeding when compared to the wildtype littermate controls. We also found that deletion of Jak2 in dendritic cells reduced the salt‐induced expression of ENaCγ, and serum/glucocorticoid regulated kinase 1 (SGK1) in CD11c+ cells. Following high salt feeding, mice lacking Jak2 in DCs exhibited less renal infiltration of effector memory T cells (TEM), less aortic infiltration of CD11c+ cells with less expression of CD86, and less production of IsoLGs and IL1‐beta. These results indicate that dendritic cell Jak2 plays an important role in salt‐sensitive hypertension through an ENaC‐dependent mechanism.