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
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