High salt activates NLRP3 inflammasome in antigen presenting cells via ENaC to promote salt‐sensitive hypertension

Abstract

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 InformationK01HL130497, R01HL147818, T32HL144446