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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.

Summary Background Endovascular renal denervation reduces blood pressure in patients with mild-to-moderate hypertension, but its efficacy in patients with true resistant hypertension has not been shown. We aimed to assess the efficacy and safety of endovascular ultrasound renal denervation in patients with hypertension resistant to three or more antihypertensive medications. Methods In a randomised, international, multicentre, single-blind, sham-controlled trial done at 28 tertiary centres in the USA and 25 in Europe, we included patients aged 18–75 years with office blood pressure of at least 140/90 mm Hg despite three or more antihypertensive medications including a diuretic. Eligible patients were switched to a once daily, fixed-dose, single-pill combination of a calcium channel blocker, an angiotensin receptor blocker, and a thiazide diuretic. After 4 weeks of standardised therapy, patients with daytime ambulatory blood pressure of at least 135/85 mm Hg were randomly assigned (1:1) by computer (stratified by centres) to ultrasound renal denervation or a sham procedure. Patients and outcome assessors were masked to randomisation. Addition of antihypertensive medications was allowed if specified blood pressure thresholds were exceeded. The primary endpoint was the change in daytime ambulatory systolic blood pressure at 2 months in the intention-to-treat population. Safety was also assessed in the intention-to-treat population. This study is registered with ClinicalTrials.gov , NCT02649426 . Findings Between March 11, 2016, and March 13, 2020, 989 participants were enrolled and 136 were randomly assigned to renal denervation (n=69) or a sham procedure (n=67). Full adherence to the combination medications at 2 months among patients with urine samples was similar in both groups (42 [82%] of 51 in the renal denervation group vs 47 [82%] of 57 in the sham procedure group; p=0·99). Renal denervation reduced daytime ambulatory systolic blood pressure more than the sham procedure (−8·0 mm Hg [IQR –16·4 to 0·0] vs –3·0 mm Hg [–10·3 to 1·8]; median between-group difference –4·5 mm Hg [95% CI –8·5 to –0·3]; adjusted p=0·022); the median between-group difference was –5·8 mm Hg (95% CI –9·7 to –1·6; adjusted p=0·0051) among patients with complete ambulatory blood pressure data. There were no differences in safety outcomes between the two groups. Interpretation Compared with a sham procedure, ultrasound renal denervation reduced blood pressure at 2 months in patients with hypertension resistant to a standardised triple combination pill. If the blood pressure lowering effect and safety of renal denervation are maintained in the long term, renal denervation might be an alternative to the addition of further antihypertensive medications in patients with resistant hypertension. Funding ReCor Medical.

Summary Background Early studies suggest that radiofrequency-based renal denervation reduces blood pressure in patients with moderate hypertension. We investigated whether an alternative technology using endovascular ultrasound renal denervation reduces ambulatory blood pressure in patients with hypertension in the absence of antihypertensive medications. Methods RADIANCE-HTN SOLO was a multicentre, international, single-blind, randomised, sham-controlled trial done at 21 centres in the USA and 18 in Europe. Patients with combined systolic–diastolic hypertension aged 18–75 years were eligible if they had ambulatory blood pressure greater than or equal to 135/85 mm Hg and less than 170/105 mm Hg after a 4-week discontinuation of up to two antihypertensive medications and had suitable renal artery anatomy. Patients were randomised (1:1) to undergo renal denervation with the Paradise system (ReCor Medical, Palo Alto, CA, USA) or a sham procedure consisting of renal angiography only. The randomisation sequence was computer generated and stratified by centres with randomised blocks of four or six and permutation of treatments within each block. Patients and outcome assessors were blinded to randomisation. The primary effectiveness endpoint was the change in daytime ambulatory systolic blood pressure at 2 months in the intention-to-treat population. Patients were to remain off antihypertensive medications throughout the 2 months of follow-up unless specified blood pressure criteria were exceeded. Major adverse events included all-cause mortality, renal failure, an embolic event with end-organ damage, renal artery or other major vascular complications requiring intervention, or admission to hospital for hypertensive crisis within 30 days and new renal artery stenosis within 6 months. This study is registered with ClinicalTrials.gov, number NCT02649426. Findings Between March 28, 2016, and Dec 28, 2017, 803 patients were screened for eligibility and 146 were randomised to undergo renal denervation (n=74) or a sham procedure (n=72). The reduction in daytime ambulatory systolic blood pressure was greater with renal denervation (−8·5 mm Hg, SD 9·3) than with the sham procedure (−2·2 mm Hg, SD 10·0; baseline-adjusted difference between groups: −6·3 mm Hg, 95% CI −9·4 to −3·1, p=0·0001). No major adverse events were reported in either group. Interpretation Compared with a sham procedure, endovascular ultrasound renal denervation reduced ambulatory blood pressure at 2 months in patients with combined systolic–diastolic hypertension in the absence of medications. Funding ReCor Medical.

A class of mutants of Paramecium tetraurelia has been isolated by adding a critical amount of K + to mutagenized, autogamized cultures. Adding 35 mM K + to the medium kills the wild type. The mutants can grow at 35 mM K + and some survive an addition of up to 80 mM K + . Thirty-three lines of such mutants have been studied. These mutants have few or no behavioral abnormalities in culture medium. Paramecia can “adapt.” Hildebrand and Dryl showed that P. caudatum incubated in 15 mM K + for over 15 min fail to give avoiding reactions when challenged with Ba 2+ or a thermal gradient [Hildebrand, E. & Dryl, S. (1976) Bioelectrochem. Bioenerg . 3, 543-544]. We have observed a similar phenomenon in wild-type P. tetraurelia . However, the K + -resistant mutants show little or no such “adaptation,” i.e., they perform avoiding reactions when challenged with Ba 2+ after prolonged incubation in 15 mM K + . The K + resistance and the lack of “adaptation” are strictly correlated. Because all 33 lines of mutants show this correlation, “adaptation” and killing by K + must share a mechanism. This mechanism may be studied by finding the basis of the K + resistance in the mutants. The possible ionic bases of “adaptation” are discussed.

Publisher Summary This chapter reviews the evidence that the Shaker locus in Drosophila is the site for the structural gene of a K+ channel. It discusses the strategies to be used for the molecular cloning of the Shaker locus. The recently isolated hybrid dysgenesis-induced Shaker mutants, which can be useful in the initial cloning and subsequent analysis of DNA from the Shaker locus, are described. The molecular studies of K+ channels are important because these channels play important roles in the control of neuronal activity and synaptic efficacy. The genetics of Drosophila and mutations of the Shaker locus offer an alternative approach for cloning K+ channels in the absence of high-affinity toxins or antibodies against K+ channels. In addition to providing a starting point for cloning, dysgenesis-induced Shaker mutants can also supply abundant new mutations that are considered useful in later molecular analysis. Genetic analyses using electrophysiological assays including voltage clamping provide strong evidence that the Shaker locus contains the structural gene for a K+ channel, the A channel.