Ashley Pitzer1, Fernando Elijovich1, Cheryl L Laffer1, Lale A Ertuglu2, Melis Sahinoz2, Mohammad Saleem1, Jaya Krishnan1, Thanvi Dola1, Luul A Aden1, Quanhu Sheng3, Michael A Raddatz4,5, Celestine Wanjalla6, Suman Pakala6, Sean S Davies7,8, David M Patrick1,9,10, Valentina Kon11, T Alp Ikizler, Thomas Kleyman12, Annet Kirabo1,13. 1. Division of Clinical Pharmacology (A.P., F.E., C.L.L., M. Saleem, J.K., T.D., L.A.A., D.M.P., A.K.), Vanderbilt University Medical Center, Nashville, TN. 2. Division of Nephrology (L.A.E., M. Sahinoz), Vanderbilt University Medical Center, Nashville, TN. 3. Department of Medicine, Department of Biostatistics (Q.S.), Vanderbilt University Medical Center, Nashville, TN. 4. Medical Scientist Training Program, Vanderbilt University, Nashville, TN (M.A.R.). 5. Department of Medicine, University of California, Los Angeles (M.A.R.). 6. Division of Infectious Diseases, Department of Internal Medicine (C.W., S.P.), Vanderbilt University Medical Center, Nashville, TN. 7. Department of Pharmacology (S.S.D.), Vanderbilt University, Nashville, TN. 8. Vanderbilt Institute of Chemical Biology (S.S.D.), Vanderbilt University, Nashville, TN. 9. Division of Cardiovascular Medicine (D.M.P.), Vanderbilt University Medical Center, Nashville, TN. 10. Department of Pediatrics (D.M.P.) Vanderbilt University Medical Center, Nashville, TN. 11. Department of Veterans Affairs, Nashville, TN (V.K.). 12. Departments of Medicine, Cell Biology, Pharmacology and Chemical Biology, University of Pittsburgh, PA (T.K.). 13. Vanderbilt Institute for Infection' Immunology' and Inflammation, Vanderbilt University Medical Center, Nashville, TN (A.K.).
Abstract
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: 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.
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