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Evidence from nonhuman animal models demonstrates an important role for immune cells in hypertension, but immune cell changes in human hypertension are less clear. Using mass cytometry, we demonstrate novel and selective reductions in CCR10(+) regulatory T cells (Tregs) and PD-1(+)CD57(−)CD8(+) memory T cells. RNA sequencing reveals that CCR10(+) Tregs exhibit gene expression changes consistent with enhanced immunosuppressive function. In addition, CITE-Seq demonstrates that PD-1(+)CD57(−)CD8(+) memory T cells exhibit features of T-cell exhaustion. Taken together, these results provide novel evidence for decreases in anti-inflammatory and/or hypofunctional T-cell populations that may contribute to enhanced inflammation in human hypertension.

Even though it has been more than a decade since renal denervation (RDN) was first used to treat hypertension and an intense effort on researching this therapy has been made, it is still not clear how RDN fits into the antihypertensive arsenal. There is no question that RDN lowers blood pressure (BP), it does so to an extent at best corresponding to one antihypertensive drug. The procedure has an excellent safety record. However, it remains clinically impossible to predict whose BP responds to RDN and whose does not. Long-term efficacy data on BP reduction are still unconvincing despite the recent results in the SPYRAL HTN-ON MED trial; experimental studies indicate that reinnervation is occurring after RDN. Although BP is an acceptable surrogate endpoint, there is complete lack of outcome data with RDN. Clear indications for RDN are lacking although patients with resistant hypertension, those with documented increase in activity of the sympathetic system and perhaps those who desire to take fewest medication may be considered. ispartof: JOURNAL OF HYPERTENSION vol:40 issue:10 pages:1859-1875 ispartof: location:Netherlands status: published

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.

Significance: Salt sensitivity of blood pressure (SSBP) is an independent risk factor for mortality and morbidity due to cardiovascular disease, and disproportionately affects blacks and women. Several mechanisms have been proposed, including exaggerated activation of sodium transporters in the kidney leading to salt retention and water. Recent Advances: Recent studies have found that in addition to the renal epithelium, myeloid immune cells can sense sodium via the epithelial Na(+) channel (ENaC), which leads to activation of the nicotinamide adenine dinucleotide phosphate oxidase enzyme complex, increased fatty acid oxidation, and production of isolevuglandins (IsoLGs). IsoLGs are immunogenic and contribute to salt-induced hypertension. In addition, aldosterone-mediated activation of ENaC has been attributed to the increased SSBP in women. The goal of this review is to highlight mechanisms contributing to SSBP in blacks and women, including, but not limited to increased activation of ENaC, fatty acid oxidation, and inflammation. Critical Issues: A critical barrier to progress in management of SSBP is that its diagnosis is not feasible in the clinic and is limited to expensive and laborious research protocols, which makes it difficult to investigate. Yet without understanding the underlying mechanisms, this important risk factor remains without treatment. Future Directions: Further studies are needed to understand the mechanisms that contribute to differential blood pressure responses to dietary salt and find feasible diagnostic tools. This is extremely important and may go a long way in mitigating the racial and sex disparities in cardiovascular outcomes. Antioxid. Redox Signal. 35, 1477–1493.

Salt sensitivity of blood pressure is an independent risk factor for cardiovascular mortality not only in hypertensive but also in normotensive adults. The diagnosis of salt sensitivity of blood pressure is not feasible in the clinic due to lack of a simple diagnostic test, making it difficult to investigate therapeutic strategies. Most research efforts to understand the mechanisms of salt sensitivity of blood pressure have focused on renal regulation of sodium. However, salt retention or plasma volume expansion is not different between salt-sensitive and salt-resistant individuals. In addition, over 70% of extracellular fluid is interstitial and, therefore, not directly controlled by renal salt and water excretion. We discuss in this review how the seminal work by Harry Goldblatt paved the way for our attempts at understanding the mechanisms that underlie immune activation by salt in hypertension. We describe our findings that sodium, entering antigen-presenting cells via an epithelial sodium channel, triggers a PKC (protein kinase C)- and SGK1 (serum/glucocorticoid kinase 1)-stimulated activation of nicotinamide adenine dinucleotide phosphate oxidase, which, in turn, enhances lipid oxidation with generation of highly reactive isolevuglandins. Isolevuglandins adduct to proteins, with the potential to generate degraded peptide neoantigens. Activated antigen-presenting cells increase production of the TH17 polarizing cytokines, IL (interleukin)-6, IL-1β, and IL-23, which leads to differentiation and proliferation of IL-17A producing T cells. Our laboratory and others have shown that this cytokine contributes to hypertension. We also discuss where this sodium activation of antigen-presenting cells may occur in vivo and describe the multiple experiments, with pharmacological antagonists and knockout mice that we used to unravel this sequence of events in rodents. Finally, we describe experiments in mononuclear cells obtained from normotensive or hypertensive volunteers, which confirm that analogous processes of salt-induced immunity take place in humans.

Salt sensitivity of blood pressure is an independent cardiovascular risk factor as powerful as hypertension, yet it goes untreated due to the lack of a feasible diagnostic tool. We previously found that sodium enters antigen-presenting cells (APCs) via ENaC, leading to the formation of isolevuglandins (IsoLGs). IsoLGs are highly reactive oxidative products of fatty acids and act as neoantigens to trigger inflammation in hypertension. We found that activation of APCs via IsoLG is highly variable in humans. Thus, we hypothesized that this variability in IsoLG-mediated APC activation contributes to the variability observed in human salt sensitivity of blood pressure and that IsoLGs can be used as a diagnostic tool. We measured systolic blood pressure (SBP) and IsoLG-containing APCs (dendritic cells [DC], classical, intermediate and non-classical monocytes) by flow cytometry in 14 hypertensive subjects who were off therapy for 2 weeks, before (B) and after in-patient 24 hr salt loading (HI, 460 mmol Na + ) and salt depletion (LO, 10 mmol Na + and furosemide 40 mg x 3). Salt sensitivity was assessed by ΔSBP (LO minus HI). Median age was 49, with 5 females, median BMI (IQR) of 29.8 (25.2, 39.5) kg/m 2 and screening SBP of 135.1 (130, 144.9) mmHg. The median urinary Na + excretions (IQR) were 169.6 (125, 203.3), 360.9 (324.1, 421.2) and 54.8 (36.8, 76) mmol/24h at baseline, HI and LO days, respectively. Baseline percentages of classical and non-classical monocytes with IsoLGs were positively correlated with ΔSBP (r=0.64, p=0.01 and r=0.71, p

BACKGROUND: There are limited and nonconcordant data on the rapidity and safety of blood pressure response to clonidine in the setting of asymptomatic severe hypertension. We evaluated the blood pressure response to clonidine in hospitalized patients with asymptomatic severe hypertension. METHODS: We performed a review of hospitalized, noncritically ill patients receiving clonidine within 6 hours of developing asymptomatic severe hypertension (systolic blood pressure [SBP] >180 or diastolic blood pressure [DBP] >110 mm Hg in the absence of acute hypertension-mediated target organ damage). The incidence of mean arterial pressure (MAP) reduction by ≥30% at 4 hours after clonidine was the primary endpoint. RESULTS: We identified 200 relevant patient encounters (median age 63 years, 48.5% women). Median time to clonidine following asymptomatic severe hypertension was 2.8 hours. A total of 20 (10%) patients had ≥30% MAP reduction within 4 hours after clonidine, and 32 (16%) patients had ≥30% reduction in either SBP, DBP, or MAP. Older age, female sex, and preexisting vascular disease were associated with ≥30% MAP reductions (P < 0.05). Only patient sex and clonidine dose of 0.3 mg were significant in multivariable models. There were 14 adverse events observed within 24 hours of administration of clonidine; most (9) were acute kidney injury. There were no ischemic (myocardial, cerebrovascular) events. CONCLUSIONS: A substantial minority of hospitalized patients with asymptomatic severe hypertension experience precipitous blood pressure decline with clonidine, and though blood pressure declines more precipitously in women and those receiving higher doses (0.3 mg specifically), the response to clonidine is generally not predictable on clinical grounds.

Background: High Na+ stimulates antigen-presenting cells (APCs) in an ENaC dependent manner, with formation of isolevuglandin (isoLG) adducts (neoantigen peptides) that promote T cell activation and salt sensitive (SS) hypertension in rodents. Methods: We studied this pathway in 9 subjects with essential hypertension who discontinued anti-hypertensive therapy for 2 weeks. Their SS was assessed by 24-hrs of salt loading (460 mmoL) and salt depletion (10 mmoL/24 hr, plus furosemide 40 mg x 3). Muscle and skin Na + were measured at baseline (BA) by 23 Na magnetic resonance imaging (NaMRI). The % of APCs containing isoLG adducts (flow cytometry), urine and serum electrolytes and epoxyeicosatrienoic acids (EETs 8-9, 11-12 and 14-15) were measured at BA, after salt-loading (HI) and after salt-depletion (LO). Results: Age was 54 years (48-56), with 23% female, BMI 30 kg/m 2 (28-40) and screening SBP 136 mmHg (120-144), and DBP 85 mmHg (75-99). BA 24-hr urine Na + excretion was 178 (143-212) mmoL, Hi 392 (229-421) and LO 27 (25-29). SBP response to salt-depletion varied from -13.8 to +5.6 mmHg. Muscle Na+ correlated with duration of hypertension (r=0.73, p Conclusions: Muscle Na+ increases with duration of hypertension and correlates with severity of BP elevation. Changes in APC isoLGs due to Na+ loading or depletion seem to be a biomarker of SS of BP in humans. Relations between urine EETs and ΔEETs with APC isoLGs and ΔisoLGs suggest that EETs might be inhibitors of APC ENaC as they are of renal ENaC. Relationships between isoLGs and urine but not plasma EETs suggest that activation of APCs by high salt may occur in the hyperosmolar renal medulla.

Emerging evidence from animal models has demonstrated the importance of multiple innate and adaptive immune cells in hypertension. We hypothesized that the abundance and phenotype of circulating immune cell subsets are altered in human hypertension. To test this, we performed high dimensional single cell profiling of human peripheral blood mononuclear cells using mass cytometry. Unsupervised computational analysis revealed a 40% decrease in CD8 + memory T cells in hypertensive individuals. Using Phenograph to identify subsets of these cells revealed a selective 60% decrease in PD-1 + CD8 + memory T cells in hypertension. This observation was confirmed in a validation cohort using flow cytometry in which PD-1 + CD8 + memory T cells were significantly decreased 44% in hypertensive compared to control individuals. To determine the phenotype of these PD-1 + CD8 + memory T cells, we performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) on four control and four hypertensive individuals. Using antibodies to identify PD-1 + and PD-1 - CD8 + memory T cells, gene set enrichment analysis of the coordinate single cell transcriptomic data revealed that PD-1 + cells exhibit over-representation of features of both immunologically active effector T cells and hypofunctional exhausted T cells. Thus, clustering analysis of PD-1 + CD8 + memory T cells was performed which demonstrated 4 distinct subclusters. One of these subclusters was decreased in hypertension and exhibited selective expression of multiple inhibitory receptors characteristic of exhausted T cells. At the protein level, this subcluster was marked by expression of the inhibitory receptor LAG3 and low levels of CD57. Combining these markers to identify PD-1 + LAG3 + CD57 - CD8 + memory T cells permitted identification of exhausted cells which demonstrated a significant 35% decrease in hypertensive compared to control individuals using flow cytometry. Taken together, these results demonstrate novel and reproducible decreases in circulating PD-1 + CD8 + memory T cells with features of exhaustion in human hypertension. These findings provide new insights into the pathogenesis of human hypertension including loss and/or re-invigoration of exhausted T cells.

Salt sensitivity of blood pressure is an independent predictor of death due to cardiovascular events. Diagnosis of salt-sensitivity is not feasible in the clinic, making it difficult to investigate therapeutic strategies. We hypothesized that NLRP3-inflammasome and IL-1β production in monocytes plays a role in salt-sensitive hypertension. We phenotyped salt-sensitivity of blood pressure using an acute inpatient Weinberger protocol of an isocaloric high salt diet and rapid intravenous salt-loading, followed by low salt diet and furosemide-induced salt-depletion. Ambulatory blood pressure was continuously monitored and averaged for the days of salt-loading and salt-depletion. Blood samples were obtained at baseline, salt-loading, and after salt-depletion. Median age was 54 years (44-55), 3 of the 5 subjects were female, screening systolic blood pressure was 140 mmHg (134-148), diastolic blood pressure was 88 mmHg (84-99), and BMI was 35 kg/m 2 (30-39). Using cell hashing and ECCITE-seq analysis, we profiled transcriptomes in multiple immune cell types using antibody-derived tags (ADTs). UMAP clustering of different cell types were identified by ADTs including monocyte markers CD14 and CD16. Interestingly, UMAP visualization of CD14+ and CD16+ clusters indicated a greater decrease in CD14+ clusters after salt-depletion in the salt-resistant subjects than the salt-sensitive; however the salt-sensitive subjects had a greater decrease in CD16+ clusters than the salt-resistant group after both salt-loading and salt-depletion. These data were confirmed using flow cytometry. Unlike in salt-resistant participants, we found that within monocyte clusters, salt-sensitivity was associated with down regulation of the inflammasome components NLRP3 (0.386 ± 1.18 vs. 0.197 ± 0.778) and IL-1β (0.858 ± 2.32 vs. 0.159 ± 0.925) following salt-depletion. Using flow cytometry, we found Δ% isoLG+ CD14+/CD16+ monocytes correlated with salt-sensitivity of blood pressure (r=0.88, 95% CI, p=0.05). These results suggest that the inflammasome and monocyte activation are dynamically regulated by dietary salt in vivo and can serve as a potential diagnostic biomarker for salt-sensitivity of blood pressure.

Background High blood pressure (BP) is associated with high-salt consumption especially in sub-Saharan Africa. Although the pressor effect of salt is viewed as a chronic effect, some studies suggest that a salty meal may increase BP immediately in some individuals, and that this effect may cause endothelial dysfunction. Therefore, the aim of our research was to study the immediate pressor response to oral salt (IPROS) and its determinants, with the expectation that a simple methodology may be devised to diagnose it in the clinic or in low-resource environments. Methods We conducted a time series trial at Livingstone Central Hospital. We present data in 127 normotensive participants who ingested 2 g of sodium chloride; their BP was monitored for 120 minutes in intervals of 10 minutes. Sociodemographic and clinical data were collected. Descriptive and inferential statistics were used for analyses of data. Results Median age was 30 years (interquartile range, 22–46 years) and 52% were female patients. An increase of ≥10 mmHg in mean arterial pressure (MAP), considered a clinically significant IPROS, was present in 62% of participants. Systolic BP 30 minutes after the salt load was a significant predictor of IPROS, avoiding the need to calculate MAP in the clinic setting. Conclusions We confirm the presence of an IPROS in a high proportion (62%) of otherwise normotensive participants. The average time course for this response was 30 minutes and its duration was sustained for the 120-minutes period of study in most of the participants. Prediction of IPROS by ∆SBP (change in systolic blood pressure) at 30 minutes allows for easy assessment of possible responder status in the clinic. Our data indicate that the IPROS to oral salt-loads in the range currently consumed by the Western world and African populations in single meals may increase the 24-hour BP load, which is a risk factor for hypertension and target organ damage. The relevance of our findings indicates the need to include dietary sodium assessment in the diagnosis, prevention, and management of high BP.

Elevated cardiovascular risk including stroke, heart failure, and heart attack is present even after normalization of blood pressure in patients with hypertension. Underlying immune cell activation is a likely culprit. Although immune cells are important for protection against invading pathogens, their chronic overactivation may lead to tissue damage and high blood pressure. Triggers that may initiate immune activation include viral infections, autoimmunity, and lifestyle factors such as excess dietary salt. These conditions activate the immune system either directly or through their impact on the gut microbiome, which ultimately produces chronic inflammation and hypertension. T cells are central to the immune responses contributing to hypertension. They are activated in part by binding specific antigens that are presented in major histocompatibility complex molecules on professional antigen-presenting cells, and they generate repertoires of rearranged T-cell receptors. Activated T cells infiltrate tissues and produce cytokines including interleukin 17A, which promote renal and vascular dysfunction and end-organ damage leading to hypertension. In this comprehensive review, we highlight environmental, genetic, and microbial associated mechanisms contributing to both innate and adaptive immune cell activation leading to hypertension. Targeting the underlying chronic immune cell activation in hypertension has the potential to mitigate the excess cardiovascular risk associated with this common and deadly disease.

Purpose of Review Salt sensitivity of blood pressure (SSBP) is an independent predictor of death due to cardiovascular events and affects nearly 50% of the hypertensive and 25% of the normotensive population. Strong evidence indicates that reducing sodium (Na+) intake decreases blood pressure (BP) and cardiovascular events. The precise mechanisms of how dietary Na+ contributes to elevation and cardiovascular disease remain unclear. The goal of this review is to discuss mechanisms of salt-induced cardiovascular disease and how the microbiome may play a role. Recent Findings The innate and adaptive immune systems are involved in the genesis of salt-induced hypertension. Mice fed a high-salt diet exhibit increased inflammation with a marked increase in dendritic cell (DC) production of interleukin (IL)-6 and formation of isolevuglandins (IsoLG)-protein adducts, which drive interferon-gamma (IFN-γ) and IL-17A production by T cells. While prior studies have mainly focused on the brain, kidney, and vasculature as playing a role in salt-induced hypertension, the gut is the first and largest location for Na+ absorption. Research from our group and others strongly suggests that the gut microbiome contributes to salt-induced inflammation and hypertension. Summary Recent studies suggest that alterations in the gut microbiome contribute to salt-induced hypertension. However, the contribution of the microbiome to SSBP and its underlying mechanisms are not known. Targeting the microbiota and the associated immune cell activation could conceivably provide the much-needed therapy for SSBP.

Introduction: Salt Sensitivity (SS) of blood pressure (BP) is an independent predictor of death due to cardiovascular disease, but its pathogenesis is poorly understood. Sodium (Na + ) is stored in the skin and muscle interstitium. This hyperosmolar Na + activates monocytes in vitro via oxidative stress with generation of isolevuglandin (isoLG) protein adducts that are immunogenic and activate the adaptive immune system. Methods: Five subjects with essential hypertension discontinued all anti-hypertensive therapy for two weeks before the study. SS was assessed by an inpatient protocol of salt loading (460 mmoL/24h) and salt depletion (10 mmoL/24h, plus furosemide 40 mg x 3). Muscle and skin Na + contents were measured at baseline (BA) by 23 Sodium magnetic resonance imaging ( 23 NaMRI). Urine and serum electrolytes, glomerular filtration rate and the % CD14 + monocytes containing isoLG adducts using flow cytometry were obtained at BA, after salt-loading (HI) and after salt-depletion (LO). All continuous data are displayed as median (interquartile range). Spearman’s correlation was used to test associations. Results: Median age was 54 years (44-55), 60% of subjects were female, screening systolic BP (SBP) was 140 mmHg (134-148), diastolic BP was 88 mmHg (84-99) and BMI was 35 kg/m 2 (30-39). SBP response to salt-depletion (salt-sensitivity index, SSI) varied from -13.8 to +1.8 mmHg. %isoLG + CD14 + cells were 48 (27-65) at BA, 55 (31-56) at HI, and 70 (33-72) at LO (p=0.594, by the Kruskal-Wallis test). The correlation between SSI and delta (Δ) %isoLG LO minus HI, was 0.86, [95% confidence interval (CI), -0.07-0.99] which may suggest conclusively as we gather more data that the greater the SSI, the larger the decrease in isoLGs by salt depletion. Muscle Na + content correlated with 24h urine Na + (BA) (r=0.90, 95% CI, 0.11-0.99), however, the correlation with BP, SSI or isoLGs was inconclusive, potentially due to the small sample size. Skin Na + content correlated with baseline %CD14IsoLG + (r=0.91; 95% CI, 0.12-0.99). Conclusions: Na + intake is a component of the determinants of muscle Na + . Skin Na + is associated with increased isoLGs in monocytes, a marker of immune cell activation. Variability in ΔCD14isoLG may serve as a biomarker for SS of BP in humans.

Aims Prior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro. Methods and results To study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments. Exposure of human monocytes to elevated Na+ex vivo caused a co-ordinated response involving isolevuglandin (IsoLG)-adduct formation, acquisition of a dendritic cell (DC)-like morphology, expression of activation markers CD83 and CD16, and increased production of pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-6, and IL-1β. High salt also caused a marked change in monocyte gene expression as detected by RNA sequencing and enhanced monocyte migration to the chemokine CC motif chemokine ligand 5. NADPH-oxidase inhibition attenuated monocyte activation and IsoLG-adduct formation. The increase in IsoLG-adducts correlated with risk factors including body mass index, pulse pressure. Monocytes exposed to high salt stimulated IL-17A production from autologous CD4+ and CD8+ T cells. In addition, to evaluate the effect of salt in vivo, monocytes and T cells isolated from humans were adoptively transferred to immunodeficient NSG mice. Salt feeding of humanized mice caused monocyte-dependent activation of human T cells reflected by proliferation and accumulation of T cells in the bone marrow. Moreover, we performed a cross-sectional study in 70 prehypertensive subjects. Blood was collected for flow cytometric analysis and 23Na magnetic resonance imaging was performed for tissue sodium measurements. Monocytes from humans with high skin Na+ exhibited increased IsoLG-adduct accumulation and CD83 expression. Conclusion Human monocytes exhibit co-ordinated increases in parameters of activation, conversion to a DC-like phenotype and ability to activate T cells upon both in vitro and in vivo sodium exposure. The ability of monocytes to be activated by sodium is related to in vivo cardiovascular disease risk factors. We therefore propose that in addition to the kidney and vasculature, immune cells like monocytes convey salt-induced cardiovascular risk in humans., Graphical Abstract

A 30-year-old man, who had moved to the United Kingdom from South Asia, was referred to the renal clinic with nephrotic syndrome. He had recently been diagnosed with systemic lupus erythematosus (SLE) after presenting to the rheumatology clinic with joint pain, skin rash, and pleuritic chest pain and fulfilling 8 of 17 SLICC (Systemic Lupus International Collaborating Clinics) diagnostic criteria.1 His immunology was in keeping with active SLE: his complement levels were low, and he had antibodies against double-stranded DNA and extractable nuclear antigens (Table S1A in the online-only Data Supplement). Our patient had heavy proteinuria (3.9 g/d) and a low serum albumin (25 g/L) in keeping with the nephrotic syndrome. Although his excretory renal function was normal, he had microhematuria (3+) on urinalysis. An urgent renal tract ultrasound with Doppler revealed that he had a preexistent renal vein thrombosis for which he was anticoagulated. In the absence of any serological evidence of antiphospholipid syndrome, this was attributed to his nephrotic syndrome. He went on to have a renal biopsy. This demonstrated classes III (focal proliferative) and V (membranous) lupus nephritis (Figure 1). Figure 1. Initial glomerular histology. A , Normal glomerulus. Single arrow: Normal capillary wall. This should be a similar thickness to the tubular epithelium (arrowhead). Double arrow: Normal mesangium. B , Classes III (focal proliferative) and V (membranous) lupus nephritis. Single arrow: Thickened capillary wall. Double arrow: Focal proliferation. At presentation, his blood pressure (BP) was 126/88 mm Hg in the absence of antihypertensive treatment. Although this lay within both UK and US recommended guidelines, …

Hypertension is the leading risk factor for morbidity and mortality worldwide. Emerging evidence in animal models demonstrates the importance of a variety of innate and adaptive immune cells in hypertension. We hypothesized that the abundance and phenotype of specific circulating immune cell subsets is altered in human hypertension reflecting disease pathophysiology. We performed unbiased high dimensional, single cell profiling of peripheral blood mononuclear cells in humans with a panel of 31 cell surface markers using mass cytometry. Unsupervised computational analysis from 11 control and 10 hypertensive individuals matched for age, gender, race, and body mass index revealed consistent increases in a novel memory helper T cell subset in hypertension. Manual two dimensional gating revealed that this CD4 + CD45RO + CD62L + CCR7 - CD161 lo memory cell population is nearly 2-fold increased in hypertensive subjects (1.0 vs 1.9%). As an alternative to starting with unsupervised analysis, manual gating for major known immune cell populations revealed a 40% decrease in memory cytotoxic T cells and a 29% decrease in regulatory T cells in hypertensive individuals (3.2 vs 1.9% and 1.3 vs 0.93%, respectively). Unsupervised analysis of the cellular subsets of these populations using Phenograph revealed a selective 63% decrease in PD-1 + memory cytotoxic T cells and 49% decrease in CCR10 + regulatory T cells in hypertension (0.67 vs 0.25% and 0.37 vs 0.19%, respectively). Taken together, these results demonstrate that in human hypertension there are increases in a novel memory helper T cell subset and decreases in PD-1 + memory cytotoxic and CCR10 + regulatory T cell populations. These findings may provide new insights into hypertension pathogenesis and potential therapeutic targets.

We and others have demonstrated that interleukin-17A (IL-17A) plays a critical role in the pathogenesis of cardiovascular disease and hypertension. Patients with psoriasis, an IL-17A mediated autoimmune disease, are at elevated risk for cardiovascular events. New biologic therapies for psoriasis have been developed that target IL-17A (secukinumab) or its receptor (brodalumab), but their impact on incident hypertension and its associated complications is unknown and remain unexplored outside of short-term safety in clinical trials. Our goal is to perform a retrospective case-control study utilizing the Synthetic Derivative (Vanderbilt’s de-identified version of the electronic health record) to determine whether incident hypertension or other major adverse cardiovascular events is reduced in patients with psoriasis receiving IL-17A targeted therapies versus other therapeutics. Patients are defined as cases by 2 or more mentions of a psoriasis ICD9 or ICD10 code and at least one documented prescription for secukinumab or brodalumab. Controls are defined by the presence of 2 or more psoriasis ICD codes, no documented exposure to the drugs of interest, and a mention of “psoriasis” in their Problem List. Using these definitions, we identified 571 cases and 4484 controls in the Synthetic Derivative. Of the 571 potential cases identified, 65 were confirmed by manual review for an initial validation study. Of the manually confirmed cases, 22 (34%) were male and 43 (66%) were female. Manually reviewed cases had a mean record length of 5.0 years with an average of 29.9 discrete ICD mentions. Twenty-three cases (35%) had an ICD code for hypertension, which predated the first documented psoriasis code in eight patients. Thus, we are able to use the Synthetic Derivative to identify patients with psoriasis receiving biologic therapies and assess temporal relationships relevant to hypertension. We plan to expand this study to the larger cohort identified and analyze rates of hypertension and a composite outcome of hypertension complications, including stroke, myocardial infarction, heart failure, and chronic kidney disease, in cases and controls matched for age, gender, race, and BMI.

T and B cells have been implicated in hypertension, but the mechanisms by which they produce a coordinated response is unknown. T follicular helper (Tfh) cells that produce interleukin 21 (IL21) promote germinal center (GC) B cell responses leading to immunoglobulin (Ig) production. Here we investigate the role of IL21 and Tfh cells in hypertension. In response to angiotensin (Ang) II-induced hypertension, T cell IL21 production is increased, and Il21-/- mice develop blunted hypertension, attenuated vascular end-organ damage, and decreased interleukin 17A (IL17A) and interferon gamma production. Tfh-like cells and GC B cells accumulate in the aorta and plasma IgG1 is increased in hypertensive WT but not Il21-/-mice. Furthermore, Tfh cell deficient mice develop blunted hypertension and vascular hypertrophy in response to Ang II infusion. Importantly, IL21 neutralization reduces blood pressure (BP) and reverses endothelial dysfunction and vascular inflammation. Moreover, recombinant IL21 impairs endothelium-dependent relaxation ex vivo and decreases nitric oxide production from cultured endothelial cells. Finally, we show in humans that peripheral blood T cell production of IL21 correlates with systolic BP and IL17A production. These data suggest that IL21 may be a novel therapeutic target for the treatment of hypertension and its micro- and macrovascular complications.

Monocytes play a crucial role in immune activation and the development of hypertension. Deletion of monocytes prevents experimental hypertension, and monocyte derived dendritic cells (DCs) and macrophages promote T cell activation and tissue damage. Our group recently found that monocytes exposed to hypertensive mechanical stretch convert to a pro-inflammatory phenotype denoted by expression CD14 + CD16 + , also known as intermediate monocytes. We aimed to determine whether hypertension has a direct effect on the bone marrow to activate the production and mobilization of monocytes. To study this, we initially analyzed the circulating monocytes from 15 normotensive and 12 hypertensive subjects using flow cytometry and found that hypertensive subjects have an absolute monocytosis compared to normotensives (1014 ± 113 vs. 567.5 ± 97 per uL, p < 0.05). This is associated with high numbers in both the classical CD14 + CD16 - monocytes and the non-classical CD14 low CD16 + monocytes. To further study mechanisms involved, we analyzed monocytes in the blood and bone marrow from C57BL/6 mice following two weeks of sham or Ang II infusion (490 ng/kg/min, s.c.). Similar to the findings in humans, more total monocytes were found in mice with Ang II-induced hypertension (46.7±10.1 vs 18.7±4.7 per μL, p

Hypertension is the leading risk factor for global morbidity and mortality. Emerging evidence in animal models implicates a variety of innate and adaptive immune cells in hypertension pathogenesis, and limited studies in humans have demonstrated increased circulating memory and senescent T cells in hypertensive individuals. However, the abundance and role of specific immune cells in human hypertension remains unclear. To perform unbiased high dimensional profiling of peripheral immune cells in humans, we developed and validated a novel approach using mass cytometry to detect 31 extracellular markers at the single cell level. Initial results in a normotensive individual using this panel revealed robust identification of major circulating immune cell subsets including CD4 + cells (60% of CD45 + cells), CD8 + cells (21%), γδ T cells (6%), CD25 + CD127 lo T regulatory cells (5%), CD19 + B cells (3%), and CD11c + myeloid cells (3%). Unsupervised viSNE plots using this panel of 31 markers demonstrates grouping of distinct circulating immune cell subsets based on similarity of marker levels (Figure) . We are extending this analysis to peripheral blood mononuclear cells collected from 16 additional control and 17 hypertensive individuals matched for age, gender, race, and BMI to determine differences in the abundance of known and potentially novel circulating immune cell subsets. The combination of high dimensional mapping and unsupervised computational analysis will permit rigorous immunophenotyping of human hypertension and potentially identify novel therapeutic targets.

Aims: \ud Monocytes play an important role in hypertension. Circulating monocytes in humans exist as classical, intermediate and non-classical forms. Monocyte differentiation can be influenced by the endothelium, which in turn is activated in hypertension by mechanical stretch. We sought to examine the role of increased endothelial stretch and hypertension on monocyte phenotype and function.\ud \ud Methods and Results: \ud Human monocytes were cultured with confluent human aortic endothelial cells undergoing either 5% or 10% cyclical stretch. We also characterized circulating monocytes in normotensive and hypertensive humans. In addition, we quantified accumulation of activated monocytes and monocyte-derived cells in aortas and kidneys of mice with Angiotensin II-induced hypertension. Increased endothelial stretch enhanced monocyte conversion to CD14++CD16+ intermediate monocytes and monocytes bearing the CD209 marker and markedly stimulated monocyte mRNA expression of interleukin (IL)-6, IL-1β, IL-23, chemokine (C-C motif) ligand 4 and tumor necrosis factor α. STAT3 in monocytes was activated by increased endothelial stretch. Inhibition of STAT3, neutralization of IL-6 and scavenging of hydrogen peroxide prevented formation of intermediate monocytes in response to increased endothelial stretch. We also found evidence that nitric oxide inhibits formation of intermediate monocytes and STAT3 activation. In vivo studies demonstrated that humans with hypertension have increased intermediate and non-classical monocytes and that intermediate monocytes demonstrate evidence of STAT3 activation. Mice with experimental hypertension exhibit increased aortic and renal infiltration of monocytes, dendritic cells and macrophages with activated STAT3.\ud \ud Conclusions: \ud These findings provide insight into how monocytes are activated by the vascular endothelium during hypertension. This is likely in part due to a loss of nitric oxide signaling and increased release of IL-6 and hydrogen peroxide by the dysfunctional endothelium and a parallel increase in STAT activation in adjacent monocytes. Interventions to enhance bioavailable nitric oxide, reduce IL-6 or hydrogen peroxide production or to inhibit STAT3 may have anti-inflammatory roles in hypertension and related conditions.

Salt-sensitivity is present in 50% of all hypertensive individuals. Prior studies have focused on the roles of kidney, vasculature and sympathetic activity in salt-sensitivity but the contribution of immune cells is poorly understood. We recently found that in murine dendritic cells amiloride sensitive channels sense salt and trigger NADPH oxidase-dependent formation of isolevuglandin-(IsolG)-adducts. We tested the hypothesis that human monocytes exhibit salt-sensitivity leading to activation via IsoLG-adduct formation and this is associated with cardiovascular risk factors. In a cohort of 18 subjects, we found that the sodium intake, measured by 24 hours urine excretion (UNa) was positively correlated with plasma levels of IsolGs. We also measured accumulation of interstitial sodium in 70 subjects by Magnetic Sodium Resonance and evaluated their circulating monocytes by flow cytometry. Subjects with high skin sodium had higher levels of IsoLGs in their monocytes (24±6 vs 38±6 %, pp =0.04). To investigate the ability of monocytes to respond to salt, we cultured monocytes from 17 subjects in high salt environment (HS:190 mM NaCl) or normal media (NS:150mM NaCl) for 48 hours. In culture, 47% of the subjects respond to salt, denoted by an increase of at least 20% in IsoLG formation (NS: 1327±240 vs. HS: 2217±653, p =0.009) as well as increased expression of the activation markers CD83 and CD86. The subjects’ cardiovascular risk factors including pulse pressure, BMI, glucose and total cholesterol positively correlated with the amount of IsolGs produced (ΔHS-NS) in response to salt (r=0.51 p

Primary autonomic failure is characterized by disabling orthostatic hypotension, but at least half of these patients have paradoxical supine hypertension. Renin–angiotensin mechanisms were not initially thought to contribute to this hypertension because plasma renin activity is often undetectable in autonomic failure. Plasma aldosterone levels are normal, however, and we recently showed that plasma angiotensin II is elevated and acts at AT 1 (angiotensin type 1) receptors to contribute to hypertension in these patients. Because aldosterone and angiotensin II can also bind mineralocorticoid receptors to elevate blood pressure, we hypothesized that mineralocorticoid receptor activation plays a role in the hypertension of autonomic failure. To test this hypothesis, we determined the acute effects of the mineralocorticoid receptor antagonist eplerenone (50 mg, oral) versus placebo on supine blood pressure in a randomized, double-blind, crossover study. Medications were given at 8:00 pm with blood pressure recorded every 2 hours for 12 hours. Ten primary autonomic failure patients with supine hypertension completed this study (7 pure autonomic failure, 2 multiple system atrophy, 1 parkinson’s disease; 7 male; 70±2 years of age). Eplerenone maximally reduced supine systolic blood pressure by 32±6 mm Hg at 8 hours after administration (versus 8±10 mm Hg placebo, P =0.016), with no effect on nocturia (12-hour urine volume: 985±134 mL placebo versus 931±94 mL eplerenone, P =0.492; nocturnal weight loss: −1.19±0.15 kg placebo versus −1.18±0.15 kg eplerenone, P =0.766). These findings suggest that inappropriate mineralocorticoid receptor activation contributes to the hypertension of autonomic failure, likely independent of canonical mineralocorticoid effects, and provides rationale for use of eplerenone in these patients.

Removal of the carotid body (CB) improves animal models of hypertension (HTN) and heart failure, presumably via withdrawal of chemoreflex-induced sympathetic activation. The effect of CB tumor (CBT) resection on blood pressure (BP) in subjects with HTN is unknown. We conducted a retrospective analysis of 20 subjects with HTN (BP≥140/90 mmHg or use of antihypertensives) out of 134 who underwent CBT resection. Short-term (from 3 months before to the first reading after 30 days from surgery) and long-term (slope of the regressions on time over the entire follow up) changes in BP and heart rate (HR) were ascertained and adjusted for covariates (interval between readings, total follow up, number of readings and changes in therapy). Age and duration of HTN were 56±4 and 9±5 years. Adjusted short-term decreases in systolic (SBP: −9.9±3.1, p

We measured epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs) in 21 normotensive subjects classified as salt resistant (13) or salt sensitive (8) with an inpatient protocol of salt loading (460 mEq Na + /24 hours, HiNa) and depletion (10 mEq Na + /24 hours+furosemide 40 mg×3, LoNa). No urine EETs were detected; hence, enzyme linked innumosorbent assay 14,15-DHETs (dihydroxyeicosatrienoic acids) were considered the total converted 14,15-urine pool. We report ultra-performance liquid chromatography/tandem mass spectrometry plasma EETs, DHETs, and their sum (plasma total pool) for the 3 regioisomers (8,9-, 11,12-, 14,15-) and their sum (08,15-). In salt-resistant subjects, urine total pool was unchanged by HiNa, decreased by LoNa, and correlated with urine sodium excretion, fractional excretion of Na + , and Na + /K + ratio for the 3 days of the experiment combined ( P + /K + ratio but showed correlations between EETs, blood pressures, and catecholamines and between DHETs and aldosterone ( P + /K + ratio during LoNa only. Plasma total pool of salt-sensitive was lower than in salt-resistant subjects and did not correlate with blood pressures or aldosterone but did with catecholamines. We conclude that the urine total pool reflects a renal pool involved in regulation of natriuresis, whereas the plasma total pools are of systemic origin, uninvolved in Na + excretion, perhaps contributing to regulation of vascular tone. Data suggest that abnormalities in EETs in salt-sensitive subjects participate in their renal or vascular dysfunction, which has potential therapeutic implications.

Several studies have established a relationship between hypertension and salt intake; however the mechanisms by which salt causes hypertension are poorly understood. There is also evidence that sodium (Na+) accumulates in the interstitium with aging and hypertension in concentrations exceeding the plasma. We tested the hypothesis that increased NaCl would convert human monocytes to an inflammatory phenotype and to define mechanisms involved. We exposed monocytes from 17 human volunteers to normal physiological NaCl (NS: 150 mM/L), elevated NaCl (HS: 190 mM/L), or an equiosmoloar concentration of mannitol. Exposure of human monocytes to high salt, but not mannitol, increased formation of immunogenic isolevuglandins (isoLG) (NS: 1688±384 vs Mann:1762±429 vs HS: 2381± 635 MFI p

See related article, pp 927–932 Less than 60 years ago, 2 studies were published that when read together constitute a surrogate for the first clinical trial in hypertension research. In the first, Priscilla Kincaid-Smith et al1 described the fate of untreated malignant hypertension in 197 subjects. In the second,2 the same group reported their experience with results of treatment in 94 subjects over 7 years. The first series was not an intentional placebo-control group but simply the reflection of lack of provision of treatment at that time because epidemiological evidence about the risks of hypertension was in its infancy, and documentation that reducing blood pressure would produce any benefit was nonexistent. Experts even argued whether fibrinoid necrosis, the hallmark pathology for malignant hypertension, was caused by blood pressure elevation or by a toxic factor.3 The second series subjected patients to therapy with ganglionic blocking agents without a comparison group. Therapy produced side effects that we would call horrendous nowadays, particularly, paralysis of parasympathetic ganglia, which required additional pharmacological therapy. Medical students and trainees with the mindset and knowledge of 2017 are astonished by both aspects of this history: the lack of use of therapy for a rapidly fatal disease and the uncontrolled nature of the first intervention. However, in the context of the evidence at the time (total uncertainty about the benefit of therapy and lack of solid knowledge about risk), it is unquestionable that there was no ethical breach. Further, taking into consideration that 1-year mortality was reduced from 90% to 50% from the first series to the second, the results of these studies were instrumental for all future developments in the field of hypertension. When research on benefit of reducing blood pressure was extended to patients with nonmalignant hypertension, a proactive decision was made to …

We measured eicosatrienoic acids in 21 normotensive subjects classified as salt-resistant (SR=13) or salt-sensitive (SS=8) with the rapid protocol of Indiana University (high salt, HS=460 mEq Na/24 hrs, low-salt, LS=10 mEq Na/24 hrs + furosemide 40 mg x 3). No EETs were detected in urine; hence, ELISA 14-15 DHETs were taken to represent the total pool of this urine isoform (U-TP). Plasma total pools (8-9,11-12,14-15 and 14-15 P-TP) are the sum of EETs+DHETs (HPLC-MS), and their results were similar, so the former are reported. Analyses required log-transformation of not normally distributed data. U-TP was not changed by HS but decreased with LS (Δlog -0.47±0.19, p