Sodium Entry Research Articles

Abstract 49: Activator protein 1 (AP-1) Complex in Antigen Presenting Cells contributes to Salt-Sensitive Blood Pressure in Humans

Salt-sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular morbidity and mortality. The exact mechanism by which salt intake increases blood pressure is complex and not fully understood. We previously found that sodium entry into antigen-presenting cells (APCs) via the epithelial sodium channel (ENaC) activates proinflammatory cytokines to activate T cells and modulate salt-sensitive hypertension. The activator protein 1 (AP-1) transcriptional factors (FOS/JUN) have been implicated in activating the pro-inflammatory pathway, but its role in SSBP is unknown. We hypothesized that high salt activates the AP-1 signaling pathway and inflammation in APCs and contributes to SSBP. Our bulk RNA-sequencing data in human monocytes, demonstrated that high salt increases the expression of the AP-1 gene family, FOS (2,378.1 ± 480.7 vs 6,494.0 ± 945.5, p= 0.0009), and JUN (7,313.9 ± 984.9 vs11,370.0 ± 1,286.3, p= 0.0015) compared to normal salt-treated monocytes. In additional experiments, we enrolled patients with hypertension and phenotype them for SSBP using an established inpatient protocol of salt-loading/depletion and performed single-cell transcriptomic analyses on peripheral blood mononuclear cells (PBMCs). We observed expression of the e FOS (r= 0.5041, p= 0.0464) and JUN (r= 0.7083, p= 0.0021) genes changes in concert with blood pressure in salt-sensitive (SS) but not in salt-resistant (SR) patients. To confirm whether genes expression translates to protein expression, we cultured total splenocytes from salt-sensitive SV/129 mice for 24 hours in either normal (150 mM) or high salt media (190 mM) and performed flow cytometry. Compared to normal salt, high salt-induced a significant increase in the expression of FOS-JUN genes in monocytes of the spleen. We also adoptively transferred PBMCs from salt-sensitive (SS) and salt-resistant (SR) hypertensive individuals into immunodeficient NSG mice, treated with the high salt diet for three weeks and performed flow cytometry on immune cells of the kidney, aorta, and spleen. We found that immune cells from SS people demonstrated a higher propensity to infiltrate mouse tissues with increased expression of FOS-JUN genes than immune cells from SR people. These preliminary findings disclose the role of the AP-1 gene family in salt-sensitive hypertension and may provide a potential therapeutic target for the treatment and diagnosis of SSBP.

Abstract P177: High salt modulates Antigen Presenting Cell TNF-alpha production in an Isolevuglandin-dependent manner in Hypertensive Humans

Salt intake is a predisposing factor to high blood pressure in people with salt sensitivity, but the mechanisms are not fully understood. Previously, we found that the epithelial sodium channel facilitates sodium entry into antigen-presenting cells (APCs), leading to the production of Isolevuglandins (IsoLGs) and hypertension via proinflammatory cytokines. TNF-alpha is a major proinflammatory cytokine that is produced in response to oxidative stress. We hypothesize that high salt intake-associated increased production of IsoLGs activates TNF-alpha-dependent mechanisms to contribute to salt-sensitive hypertension. To test this hypothesis, we conducted bulk RNA sequencing on isolated human monocytes with and without high salt. We also performed in vivo studies on humans living with hypertension using the rigorous Weinberger salt loading and depletion protocol at baseline (B), salt loading (SL), and salt depletion (SD) timepoints. We found that in vitro high salt treatment increased human monocyte expression of TNF-α, TNFRSF1A and TNFRSF1B (117.09 ± 23.71 vs 357.82 ± 59.62, p=0.001; 4560 ± 430.02 vs 6189 ± 461.11, p=0.0014 and 43824.73 ± 6462.55 vs 54585.64 ± 5383.58, p=0.0336 respectively, [n=11]) when compared to normal salt. In vivo studies on 16 hypertensive subjects reveal mean blood pressures as baseline (SBP: 137.7 ± 3.1, DBP: 85±2.2, MAP 104.7±2.1 [mmHg]), salt-loading (SBP: 141.3 ± 2.9, DBP: 86.1±2.3, MAP 105.6±2.2 [mmHg]), and salt-depletion (SBP: 138.7 ± 2.9, DBP: 87±2.4, MAP 104.7±2.1 [mmHg]). Using flow cytometry, we determined the buildup of Isolevuglandin adducts in APCs as follows: B: 57.6±6.4, SL: 59.8±4.6, SD: 46.0±7.1 (dendritic cells); B: 78.6±5.8, SL: 77.3±5.7, SD: 61.0±6.9 (CD14 + cells); B: 83.2±4.9, SL: 74.0±6.4, SD: 63.3±8.2 (CD14 + 16 + cells) and B: 63.3± 5.7, SL: 54.2±6.1, SD: 46.8±7.9 (CD16+ cells). In response to salt loading, patient plasma levels of TNF-α were B: 13.8±0.5 pg/mL, SL: 14.1±0.6 pg/mL, and SD: 15.3±1.2 pg/mL respectively. Finally, in vivo single-cell transcriptomic analysis in peripheral blood mononuclear cells revealed that in response to high salt treatment, TNF expression changes dynamically with blood pressure in salt-sensitive, but not salt-resistant patients. These preliminary findings suggest that TNF-α plays a role in salt-sensitive hypertension, possibly following IsoLG-driven reactive oxygen species generation. Further studies are needed to delineate specific dependent mechanisms by which this occurs.

Abstract P182: Antigen Presenting Cell-specific Keap1-Nrf2 pathway Mediates Salt-Sensitive Hypertension in Humans

Background: Hypertension remains the leading cause of mortality worldwide and is a primary risk factor for cardiovascular disease. Of the 1.3 billion people globally affected by hypertension, over 50% exhibit salt-sensitivity of blood pressure (SSBP), which leads to higher rates of mortality, morbidity, and kidney damage compared to those with salt-resistant hypertension. Previously our research has shown that high salt intake activates antigen-presenting cells (APCs), causing inflammation and hypertension. This pro-inflammatory response is partly driven by the epithelial sodium channel (ENaC), which facilitates sodium ion transport into APCs. While the Keap1-Nrf2 pathway is known for its role in antioxidant defense and inflammatory responses, its contribution to salt-sensitive hypertension remains unclear. We hypothesize that ENaC-dependent sodium entry triggers inflammation and activates the Sp1-MALAT1-Keap1-Nrf2 signaling axis in APCs, thereby contributing to SSBP. Method: We utilized a well-established inpatient protocol to phenotype participants for salt sensitivity of blood pressure. Additionally, we conducted RNA sequencing (RNAseq) on human monocytes exposed to elevated sodium levels in vitro and performed Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) analysis on peripheral blood mononuclear cells (PBMCs). Results: We found that whereas high salt did not significantly increases expression of Sp1 (5380.63 ± 359.77 vs 7414.54 ± 434.19, q=0.829), MALAT1 (86888.54 ±12200.12 vs 219429.90 ± 109994.16, q=0.717), Keap1 (1586.54 ± 132.94 vs 2203.72 ± 177.48, q=0.884), it significantly increased Nrf2 expression (5080.36 ± 416.64 vs 5443.54 ± 432.78, q=0.019) in RNAseq analysis of human high salt compared to normal salt treated monocyte. Furthermore, the results of the CITE-seq experiment indicated that changes in the expression of the Sp1 correlate with SSBP (r=0.5205, p=0.038). Conclusion: These preliminary findings reveal a physiological link between inflammation and salt-sensitive hypertension through the Sp1-MALAT1-Keap1-Nrf2 signaling axis in APCs. The activation of APCs mediated by Sp1 could potentially serve as a diagnostic marker for salt sensitivity in blood pressure.

Neuroprotective effects of MK-801 against cerebral ischemia reperfusion

Introduction& Objective: Cerebral ischemia/reperfusion (I/R) injury, the second cause of death globally, involves increased NMDA receptor activity leading to neuronal damage due to excessive sodium and calcium ion entry. Therefore, targeting NMDA receptor may potentially reduce cell death induced by brain injury. Our study aimed to investigate the role of NMDA receptors in hippocampal neuronal activity induced by I/R. MethodsIn this study, Wistar rats were divided into four groups: sham, I/R, I/R + MK801, and I/R + NMDA. Cerebral I/R injury was induced by temporarily occluding the common and vertebral carotid arteries, followed by reperfusion. MK801 or NMDA was administered to the rats after a specific reperfusion time. Neuronal density and cell morphology in the hippocampal CA1 region were assessed using Nissl and H&E staining. The expression of BDNF, p-CREB, and c-fos was evaluated through Western blot analysis. Additionally, neuronal activity in CA1 pyramidal neurons were examined using single unit recording technique. ResultsOur results showed that cerebral I/R injury caused significant damage to CA1 pyramidal neurons compared to the sham group. However, treatment with MK-801 improved hippocampal cell survival compared to the I/R group. Furthermore, MK-801 administration in I/R rats increased BDNF, c-fos, and p-CREB levels while decreasing cleaved caspase-3 activity compared to the I/R group. Additionally, electrophysiological data showed that MK-801 increased firing rates of CA1 pyramidal neurons during the reperfusion phase. ConclusionMK-801 shows promise as a therapeutic agent for cerebral I/R injury by enhancing cell survival, upregulating neuroplasticity factors, and increasing firing rates of CA1 pyramidal neurons. It exerts a specific protective effect against cerebral I/R injury.

Myeloid Cell Glucocorticoid, Not Mineralocorticoid Receptor Signaling, Contributes to Salt-Sensitive Hypertension in Humans via Cortisol.

Salt sensitivity of blood pressure (SSBP) is an independent risk factor for cardiovascular morbidity and mortality, yet the etiology is poorly understood. We previously found that serum/glucocorticoid-regulated kinase 1 (SGK1) and epoxyeicosatrienoic acids (EETs) regulate epithelial sodium channel (ENaC)-dependent sodium entry into monocyte-derived antigen-presenting cells (APCs) and activation of NADPH oxidase, leading to the formation of isolevuglandins (IsoLGs) in SSBP. Whereas aldosterone via the mineralocorticoid receptor (MR) activates SGK1 leading to hypertension, our past findings indicate that levels of plasma aldosterone do not correlate with SSBP, and there is little to no MR expression in APCs. Thus, we hypothesized that cortisol acting via the glucocorticoid receptor (GR), not the MR in APCs mediates SGK1 actions to induce SSBP. We performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) analysis on peripheral blood mononuclear cells of humans rigorously phenotyped for SSBP using an inpatient salt loading/depletion protocol to determine expression of MR, GR, and SGK1 in immune cells. In additional experiments, we performed bulk transcriptomic analysis on isolated human monocytes following in vitro treatment with high salt from a separate cohort. We then measured urine and plasma cortisol, cortisone, renin, and aldosterone. Subsequently, we measured the association of these hormones with changes in systolic, diastolic, mean arterial pressure and pulse pressure as well as immune cell activation via IsoLG formation. We found that myeloid APCs predominantly express the GR and SGK1 with no expression of the MR. Expression of the GR in APCs increased after salt loading and decreased with salt depletion in salt-sensitive but not salt-resistant people and was associated with increased expression of SGK1. Moreover, we found that plasma and urine cortisol/cortisone but not aldosterone/renin correlated with SSBP and APCs activation via IsoLGs. We also found that cortisol negatively correlates with EETs. Our findings suggest that renal cortisol signaling via the GR but not the MR in APCs contributes to SSBP via cortisol. Urine and plasma cortisol may provide an important currently unavailable feasible diagnostic tool for SSBP. Moreover, cortisol-GR-SGK1-ENaC signaling pathway may provide treatment options for SSBP.

Mechanical Stress Induces Sodium Entry and Osmoprotective Responses in Murine Synovial Fibroblasts.

Osteoarthritis (OA) is a multifactorial disease depending on molecular, genetic, and environmental factors like mechanical strain. Next to the cartilage and the subchondral bone, OA also affects the synovium, which is critically involved in the maintenance of joint homeostasis. As there is a correlation between the extracellular sodium content in the knee joint and OA, this study investigates the impact of sodium on OA-associated processes like inflammation and bone remodeling without and with mechanical loading in synovial fibroblasts. For that purpose, murine synovial fibroblasts from the knee joint were exposed to three different extracellular sodium chloride concentrations (-20 mM, ±0 mM and +50 mM NaCl) in the absence or presence of compressive or intermittent tensile strain. In addition to the intracellular Na+ content and gene expression of the osmoprotective transcription factor nuclear factor of activated T cells 5 (Nfat5), the gene and protein expression of inflammatory mediators (interleukin-6 (IL6), prostaglandin endoperoxide synthase-2 (Ptgs2)/prostaglandin E2 (PGE2)), and factors involved in bone metabolism (receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG)) were analyzed by qPCR and ELISA. Mechanical strain already increased intracellular Na+ and Nfat5 gene expression at standard salt conditions to levels obtained by exposure to increased extracellular Na+ content. Both high salt and compressive strain resulted in elevated IL6 and PGE2 release. Intermittent tensile strain did not increase Il6 mRNA expression or IL6 protein secretion but triggered Ptgs2 expression and PGE2 production. Increased extracellular Na+ levels and compressive strain increased RANKL expression. In contrast, intermittent tension suppressed RANKL expression without this response being subject to modification by extracellular sodium availability. OPG expression was only induced by compressive strain. Changes in extracellular Na+ levels modified the inflammatory response and altered the expression of mediators involved in bone metabolism in cells exposed to mechanical strain. These findings indicate that Na+ balance and Nfat5 are important players in synovial fibroblast responses to mechanical stress. The integration of Na+ and Na+-dependent signaling will help to improve the understanding of the pathogenesis of osteoarthritis and could lead to the establishment of new therapeutic targets.

Tmprss2 maintains epithelial barrier integrity and transepithelial sodium transport.

The mouse cortical collecting duct cell line presents a tight epithelium with regulated ion and water transport. The epithelial sodium channel (ENaC) is localized in the apical membrane and constitutes the rate-limiting step for sodium entry, thereby enabling transepithelial transport of sodium ions. The membrane-bound serine protease Tmprss2 is co-expressed with the alpha subunit of ENaC. αENaC gene expression followed the Tmprss2 expression, and the absence of Tmprss2 resulted not only in down-regulation of αENaC gene and protein expression but also in abolished transepithelial sodium transport. In addition, RNA-sequencing analyses unveiled drastic down-regulation of the membrane-bound protease CAP3/St14, the epithelial adhesion molecule EpCAM, and the tight junction proteins claudin-7 and claudin-3 as also confirmed by immunohistochemistry. In summary, our data clearly demonstrate a dual role of Tmprss2 in maintaining not only ENaC-mediated transepithelial but also EpCAM/claudin-7-mediated paracellular barrier; the tight epithelium of the mouse renal mCCD cells becomes leaky. Our working model proposes that Tmprss2 acts via CAP3/St14 on EpCAM/claudin-7 tight junction complexes and through regulating transcription of αENaC on ENaC-mediated sodium transport.

Abstract 19150: DUSP1 is Associated With Map-Kinase Signaling in Antigen Presenting Cells Contributing to Salt-Sensitivity of Blood Pressure

Salt sensitivity of blood pressure, a pathological response characterized by a marked increase in blood pressure following salt intake, acts independently of hypertension as a risk factor for cardiovascular morbidities. Our recent research has elucidated a mechanism of sodium entry into antigen presenting cells through epithelial sodium channels (ENaC), which leads to the formation of neoantigen presenting isolevuglandin protein adducts via oxidative stress. Reactive oxygen species present in the cell may impact inflammation response as well as other intracellular pathways, chiefly the MAPK pathway. Previous studies have established an inhibitory connection between the protein-coding phosphatase DUSP1 and MAPKs. We hypothesize that DUSP1 acts as a negative regulator of MAPK signaling pathways involved in the immune response to salt-sensitive hypertension. To test our hypothesis, we isolated human monocytes from healthy participants (N=11) and treated them with either normal (150 mMol/L) or high (190 mMol/L) Na+ for 72 hours in vitro. Analysis of bulk RNA sequence data points to a significant upregulation of DUSP1 in the high salt environments compared to normal salt levels (4339 ± 770.1 vs 2721 ± 546.4 , p=0.0192). We also found that the high salt treatment led to increased expression of ERK1 (HS:2066 ± 184.0 NS:1560 ± 136.7 p= 0.0652), ERK2 (HS:8364 ± 669.8 NS:6256 ± 578.3 p= 0.0281), JNK (HS:1069 + 102.2 NS:792.3 ± 74.85 p= 0.0336), and p38 (HS: 4062 ± 329.0 NS:3010 ± 292.5, p= 0.0336). To study further, we enrolled 9 hypertensive participants who discontinued anti-hypertensive therapy for 2 weeks in an inpatient protocol to phenotype them through rapid salt loading and depletion. Blood pressure was recorded and blood collected at baseline, after salt loading, and after salt depletion and CITE-Seq analysis was performed on isolated PBMCs. After salt loading, we found a negative correlation between both p38 expression and pulse pressure (r= -0.7564 p= 0.0299) and p38 expression and systolic blood pressure (r= -0.8371, p=0.0095). We found a positive correlation between DUSP1 expression and systolic blood pressure after salt depletion (r= 0.7089, p=0.0490). These results suggest that DUSP1 plays an important role in salt sensitivity of blood pressure.

SLC26A11 Inhibition Reduces Oncotic Neuronal Death and Attenuates Stroke Reperfusion Injury

Neuronal swelling is a pathological feature of stroke which contributes to the formation of cytotoxic edema. Under hypoxic condition, aberrant accumulation of sodium and chloride ions inside neurons increases osmotic pressure, leading to cell volume increase. Sodium entry pathway in neurons has been studied extensively. Here, we determine whether SLC26A11 is the major chloride entry pathway under hypoxia and could be the target for protection against ischemic stroke. In this study, electrophysiological properties of chloride current in primary cultured neurons were characterized using low chloride solution, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, and SLC26A11-specific siRNA under physiological conditions or ATP-depleted conditions. In vivo effect of SLC26A11 was evaluated on a rat stroke reperfusion model. We found that SLC26A11 mRNA in primary cultured neurons was upregulated as early as 6 h after oxygen glucose deprivation, and later, the protein level was elevated accordingly. Blockade of SLC26A11 activity could reduce chloride entry and attenuate hypoxia-induced neuronal swelling. In the animal stroke model, SLC26A11 upregulation was mainly located in surviving neurons close to the infarct core. SLC26A11 inhibition ameliorates infarct formation and improves functional recovery. These findings demonstrate that SLC26A11 is a major pathway for chloride entry in stroke, contributing to neuronal swelling. Inhibition of SLC26A11 could be a novel therapeutic strategy for stroke.

Modulation of calcium signaling “on demand” to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. Over the past two decades, major advances have been made in our understanding of the disease with the identification of germline or somatic mutations in ion channels and pumps. These mutations enhance calcium signaling, the main trigger of aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, thus mimicking some of the known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the a7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3a named a7-5HT3. Its activation by a selective agonist named PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular sodium and calcium concentrations, cell proliferation, steroid production electrophysiological properties, and gene expression. Treatment of a7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10–9 to 10–5 M) induced a significant increase in intracellular calcium concentrations. Interestingly, whereas cells were hyperpolarized in absence of stimulation (around –60 mV), PSEM-817 induced a strong depolarization, with cells rising to a membrane potential of around –10 mV. The stimulation of calcium signaling by PSEM-817 leads to an increase of CYP11B2 (encoding for aldosterone synthase) mRNA expression and aldosterone biosynthesis but did not affect cell proliferation measured at different time points (8 h, 24 h, 48 h, 72 h). Interestingly, calcium kinetic analyses revealed significant differences in response to the modulation of potassium or sodium entry into cells. Gene expression analyses of gene expression by RNAseq highlighted the activation of specific signaling pathways in response to treatment with Angiotensin II, potassium, and PSEM-817. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Abstract P2-16-07: Overexpression of the alpha subunit of the epithelial sodium channel in MDAMB231 breast cancer cells reduces cell migration and proliferation

Abstract Breast cancer is the most common cancer diagnosed in women worldwide and leads to 600,000+ deaths annually. The vast majority of breast cancer-related deaths are attributed to metastasis from the primary site in the breast to visceral organs or the brain. Therefore, understanding how these cells achieve metastasis is vital. Ion channels, such as the epithelial sodium channel (ENaC), are emerging as new targets for cancer research due to their role in regulating cell functions such as the process that allows cancer cells to undergo phenotypic changes necessary for metastasis. Ion channel dysregulation may be crucial in cancer development due to the ion channels’ potential role in controlling cancer cell characteristics such as migration and proliferation. There is limited research investigating the role ENaC may have in breast cancer. We have shown that more migratory, mesenchymal breast cancer cell lines have reduced mRNA and protein expression of ENaC compared to more epithelial breast cancer cell lines. Importantly, higher expression levels of the pore-forming alpha-ENaC subunit is correlated with an increased breast cancer survival in patients (n=1705) included in the SCAN-B data set. Bioinformatic analysis, using the SCAN-B data set, showed an increase in the expression of alpha-ENaC mRNA correlated with lower expression levels of migration and proliferation markers, suggesting ENaC has a role in breast cancer cell proliferation and migration. Therefore, this research examines the influence of stably overexpressing the pore-forming subunit of ENaC, alpha-ENaC, in breast cancer cells on their cell migration and proliferation ability. We hypothesised that increasing the expression of ENaC will restore the cells to a more epithelial state, thus reducing cell migration and proliferation. MDAMB231 breast cancer cells were engineered via a stable transfection to overexpress alpha-ENaC or to express an empty vector control. Several clones were isolated and expanded and using RTqPCR, alpha-ENaC mRNA overexpression was confirmed to consistently have 200-fold increased mRNA expression compared to control cells (n=9, p=0.0006). An increase in alpha-ENaC protein expression was also confirmed via western blotting. Using an EdU assay the alpha-ENaC overexpressing cells were shown to have a significant reduction in proliferation compared to the control cell line (n=3, p=0.0048). The alpha-ENaC overexpressing cells showed reduced migratory ability when examined in two established migration assays. In scratch wound assays, the alpha-ENaC overexpressing cells had a significant reduction in cell migration at 24 hours post scratch (n=4, p=0.0001). In Boyden chamber assays, the number of alpha-ENaC-overexpressing cells that migrated through the membrane was significantly reduced compared to the control cell line (n=4, p=0.02). Our results suggest that increased sodium entry into the cell through ENaC has a role in preventing the development of metastatic breast cancer cells, and highlights ENaC as a potential target for future breast cancer therapies. Citation Format: Sarah McQueen, Adam Ware, Heather Cunliffe, Fiona McDonald. Overexpression of the alpha subunit of the epithelial sodium channel in MDAMB231 breast cancer cells reduces cell migration and proliferation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-07.

S-13-5: UPDATES ON FAMILIAL HYPERALDSOTERONISM

The first reported (Sutherland et al. 1966) form of familial primary aldosteronism (PA) involved a father and son and was glucocorticoid-remediable (familial hyperaldosteronism type I, FH-I). In 1992, Lifton and colleagues elucidated its causation, a hybrid gene mutation composed of CYP11B1 (encoding 11beta-hydroxylase) regulatory sequences and CYP11B2 (aldosterone synthase) coding sequences causing ACTH-regulated aldosterone overproduction and hypertension, both readily controlled by low-dose glucocorticoid administration. Expression of aldosterone synthase activity within zona fasciculata (ZF) explained overproduction of hybrid steroids, 18-hydroxy- and 18-oxocortisol. Since 2011, PA-causing germline mutations in another four genes (three associated with FH) have been reported. While three encode cation channels (KCNJ5, CACNA1H and CACNA1D), one encodes an anion channel (CLCN2). FH-III is caused by inheritance of gain-of-function mutations in KCNJ5, encoding an inwardly rectifying potassium channel (GIRK-4). Depending on the exact mutation involved, phenotype varies from very severe (profound PA, hybrid steroid elevation and ZF hyperplasia and requiring bilateral adrenalectomy to control hypertension) to mild (absence of above with PA controllable with spironolactone). Most pedigrees demonstrate early-onset hypertension. Mutations reduce selectivity of GIRK-4 to potassium, permitting entry of sodium into the adrenal cortical cell, which results in cell depolarization and calcium influx, leading to increased expression of CYP11B2 and aldosterone synthesis. A gain-of-function mutation in CACNA1H, encoding a T-type voltage-gated calcium channel, was found in five of 40 subjects with early-onset PA and three relatives with early-onset hypertension (FH-IV). Adrenals appeared normal on imaging. The mutation shifts activation of zona glomerulosa (ZG) cells to a more hyperpolarized potential and slows inactivation, putatively resulting in increased calcium influx and aldosterone production. Germline mutations in CACNA1D, encoding an L-type voltage-gated calcium channel highly expressed in ZG, were identified in two of 100 individuals with early-onset PA. Both occurred de novo, and were associated with seizures and a variety of neurological abnormalities (PASNA). Both mutations cause channel activation at less depolarized membrane potentials, and one nearly abolished channel inactivation. In a large Australian family with FH-II, a mutation was found in CLCN2 encoding a chloride channel, ClC-2. CLCN2 mutations were found in another seven of 80 individuals with early-onset PA. A concurrent report found a CLCN2 mutation in one of 12 subjects with early-onset PA. PA due to CLCN2 mutations responds well to medical treatment. In the adrenal, ClC-2 localizes predominantly to ZG and mutations predispose cell depolarization, leading to calcium influx via activation of voltage-gated calcium channels and increased aldosterone production.

Aberrant inactivation of SCNN1G promotes the motility of head and neck squamous cell carcinoma.

The sodium channel epithelial 1 subunit gamma (SCNN1G) is mainly responsible for sodium entry and absorption. The dysfunction of SCNN1G has been widely studied in kidney-related diseases and chronic heart failure. However, its role in cancer remains unclear. Here, we found that SCNN1G was aberrantly downregulated in head and neck squamous cell cancer (HNSCC) tissues, which showed an efficifent diagnostic value according to the ROC curve analysis. The lower expression of SCNN1G was significantly correlated with lymphatic metastasis and a worse outcome of HNSCC patients. Ectopic overexpressing SCNN1G inhibited the invasive and migratory abilities of HNSCC cells, while knocking down SCNN1G showed an opposite effect. A positive correlation between SCNN1G and CDH1 expression was observed, which suggested that SCNN1G might impede HNSCC metastasis via strengthing cell-cell adherin. In addition, RAS signaling and ion channel transport signaling were enriched by SCNN1G in HNSCC using GSEA analysis, indicating that these signaling pathways might be the underlying mechanisms for SCNN1G as well. In addition, six sorts of immune infiltrate subtype cells were associated with SCNN1G expression. Our findings support that SCNN1G inactivation contributes to the metastasis of HNSCC. SCNN1G could serves as a valuable diagnostic and prognostic marker for HNSCC.

Effects of salinity on the nutritional quality of amaranth (Amaranthus cruentus) leaves: Case of local and resistant varieties

Climate change is a real obstacle to agriculture because of the difficulty of water supply. Farmers are trying to mitigate this problem by developing several strategies including those based on irrigation. Unfortunately, these irrigation techniques favour the entry of sodium in food crops. In response to this situation, research has been conducted in Benin to develop a salinity resistant variety of local amaranth (called line 23). The objective of this study is to determine the nutritional values of this resistant amaranth variety grown under salt stress and to compare them with the nutritional values of local amaranth grown under the same conditions. Local and resistant amaranth plants were grown in real environment and then stressed with salt concentrations of 0; 7.1 and 19.6 mmol NaCl. After harvesting, the leaves were sent to the laboratories for the determination of nutritional elements. Three replicates were done. Analysis of variance was used to compare the means of the nutritional elements assayed using JMP software (SAS Institute MC 2007). The results show a significant improvement in calcium, phosphorus, potassium and vitamin A levels in line 23 in contrast to the reference cultivar. However, the sodium content, although decreasing as the salt concentration increases, remains above acceptable limits. As for the reference cultivar, the results show an increase in the concentrations of several elements: iron, calcium, magnesium, including that of sodium, which is harmful to cardiovascular health.

Dendritic Cell Epithelial Sodium Channel in Inflammation, Salt-Sensitive Hypertension, and Kidney Damage.

Salt-sensitive hypertension is a major risk factor for cardiovascular morbidity and mortality. The pathophysiologic mechanisms leading to different individual BP responses to changes in dietary salt remain elusive. Research in the last two decades revealed that the immune system plays a critical role in the development of hypertension and related end organ damage. Moreover, sodium accumulates nonosmotically in human tissue, including the skin and muscle, shifting the dogma on body sodium balance and its regulation. Emerging evidence suggests that high concentrations of extracellular sodium can directly trigger an inflammatory response in antigen-presenting cells (APCs), leading to hypertension and vascular and renal injury. Importantly, sodium entry into APCs is mediated by the epithelial sodium channel (ENaC). Although the role of the ENaC in renal regulation of sodium excretion and BP is well established, these new findings imply that the ENaC may also exert BP modulatory effects in extrarenal tissue through an immune-dependent pathway. In this review, we discuss the recent advances in our understanding of the pathophysiology of salt-sensitive hypertension with a particular focus on the roles of APCs and the extrarenal ENaC.

DC ENaC-Dependent Inflammasome Activation Contributes to Salt-Sensitive Hypertension.

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. 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. 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. 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.

DEVELOPMENT OF AN ADRENOCORTICAL CELL MODEL OF CALCIUM SIGNALING MODULATION TO DECIPHER THE MOLECULAR MECHANISMS RESPONSIBLE FOR PRIMARY ALDOSTERONISM

Objective: Primary aldosteronism (PA) is the most frequent form of secondary hypertension. The identification of germline or somatic mutations in different genes coding for ion channels (KCNJ5, CACNA1D, CACNA1H and CLCN2) and ATPases (ATP1A1 and ATP2B3) defines PA as a channelopathy. These mutations promote activation of calcium signaling, the main trigger for aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, mimicking some of known mutations identified in PA. Design and method: We have developed an adrenocortical H295R_S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the α7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3α and named α7–5HT3. Mutations have been introduced in the ligand binding domain to allow only synthetic drugs to activate this channel receptor. Activation of α7–5HT3 by a specific drug, PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production, steroidogenic expression and electrophysiological properties. Results: Treatment of α7–5HT3 expressing cells with increasing concentration of PSEM-817 (from 10–9 to 10–5 M) induced a significant increase in intracellular calcium concentrations, similarly to potassium (12 mM) or angiotensin II (10–8 M). This stimulation of calcium signaling did not affect cell proliferation, but was responsible for an increase in CYP11B2 expression and aldosterone production after 24 h of treatment. However, while increased intracellular calcium concentrations were observed starting from 10–8 M of PSEM-817, CYP11B2 expression and aldosterone production were only affected starting from 10–7 M, suggesting a dose dependent effect. Finally, whereas cells were hyperpolarized in absence of stimulation (around -60 mV), PSEM-817 induced a strong depolarization, cells rising to a membrane potential around -10 mV. Conclusions: This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

Development of an adrenocortical cell model of calcium signaling modulation to decipher the molecular mechanisms responsible for primary aldosteronism

Primary aldosteronism (PA) is the most frequent form of secondary hypertension. The identification of germline or somatic mutations in different genes coding for ion channels and defines PA as a channelopathy. These mutations promote activation of calcium signaling, the main trigger for aldosterone biosynthesis. The objective of our work was to elucidate, using chemogenetic tools, the molecular mechanisms underlying the development of PA by modulating sodium entry into the cells, mimicking some of known mutations identified in PA. We have developed an adrenocortical H295R-S2 cell line stably expressing a chimeric ion channel receptor formed by the extracellular ligand-binding domain of the α7 nicotinic acetylcholine receptor fused to the ion pore domain of the serotonin receptor 5HT3α and named α7-5HT3. Mutations have been introduced in the ligand-binding domain to allow only synthetic drugs to activate this channel receptor. Activation of α7-5HT3 by a specific drug, PSEM-817 leads to sodium entry into the cells. This cell line was characterized in terms of intracellular calcium concentrations, cell proliferation, aldosterone production, steroidogenic expression and electrophysiological properties. Treatment of α7-5HT3 expressing cells with increasing concentration of PSEM-817 (from 10-9 to 10-5 M) induced a significant increase in intracellular calcium concentrations, similarly to potassium (12 mM) or angiotensin II (10-8 M). This stimulation of calcium signaling did not affect cell proliferation, but was responsible for an increase in CYP11B2 expression and aldosterone production after 24 h of treatment. However, while increased intracellular calcium concentrations were observed starting from 10-8 M of PSEM-817, CYP11B2 expression and aldosterone production were only affected starting from 10-7 M, suggesting a dose-dependent effect. Finally, whereas cells were hyperpolarized in absence of stimulation (around −60 mV), PSEM-817 induced a strong depolarization, cells rising to a membrane potential around −10 mV. This cell line, in which we can modulate the intracellular calcium concentration “on demand”, is a useful tool for a better understanding of the alterations of intracellular ion balance and calcium signaling in the pathophysiology of PA.

The Effects of Rhizosphere Inoculation with Pseudomonas mandelii on Formation of Apoplast Barriers, HvPIP2 Aquaporins and Hydraulic Conductance of Barley.

Pseudomonas mandelii strain IB-Ki14 has recently been shown to strengthen the apoplastic barriers of salt-stressed plants, which prevents the entry of toxic sodium. It was of interest to find out whether the same effect manifests itself in the absence of salinity and how this affects the hydraulic conductivity of barley plants. Berberine staining confirmed that the bacterial treatment enhanced the deposition of lignin and suberin and formation of Casparian bands in the roots of barley plants. The calculation of hydraulic conductance by relating transpiration to leaf water potential showed that it did not decrease in bacteria-treated plants. We hypothesized that reduced apoplastic conductivity could be compensated by the higher conductivity of the water pathway across the membranes. This assumption was confirmed by the results of the immunolocalization of HvPIP2;5 aquaporins with specific antibodies, showing their increased abundance around the areas of the endodermis and exodermis of bacteria-treated plants. The immunolocalization with antibodies against auxins and abscisic acid revealed elevated levels of these hormones in the roots of plants treated with bacteria. This root accumulation of hormones is likely to be associated with the ability of Pseudomonas mandelii IB-Ki14 to synthesize these hormones. The involvement of abscisic acid in the control of aquaporin abundance and auxins—in the regulation of and formation of apoplast barriers—is discussed.

Formation of Solonchak in the Area of the Discharged Ancient Brine Wells (Perm Krai, Russia)

The change in soil-forming processes and, as a result, in the ecosystem due to nearly 300-year long outflow of highly saline waters from ancient brine wells is considered for Perm Krai in Russia for the first time. The study area is located in the high floodplain of the Usolka River valley. Sodium-chloride ancient brine wells with mineralization of about 30 g/L flow in streams over the soil surface and enter the Usolka River. The paper considers three soil types at various distances from a saline stream. The main pedogenic processes are the sod-forming process and gleying in alluvial soils. Soils in Gleyic Fluvisols (background)—Gleyic Fluvisols (Sulfatic) (transition)—Solonchak (affected by sodium-chloride brines) series were studied in terms of occurring soil-forming processes. All studied soils are formed under hydromorphic conditions. It is evidenced by gleization. However, the morphological and physicochemical properties of the soils differ. The presence of water-soluble ions in the solonchak suggests that salinization is occurring. The sum of toxic salts was 0.94% which was several times higher than in the transition and background soils. The sodium adsorption ratio (SAR) ranged from 10.4 to 21.6, with a рН of 8.3–9.1. The saline soils were more alkaline (pH average 8.8 and 9.3) than the background soil (pH 8.1). SAR decreased significantly in the Solonchak—transition—background soil series from 16.9 to 0.7. The aforementioned observations, as well as physical properties of the saline soil, clearly show the entry of sodium into the solonchak adsorption complex, indicating the solonization process. The study area is characterized by the presence of salt-tolerant species of herbaceous plants and an obligate halophyte. Salicornia perennans Willd, a halophyte, was discovered in the wells’ discharge area at a distance of 0.2–1.5 m from saline streams. The analysis of the long-term impact of highly mineralized waters on the natural environment will aid in revealing and forecasting ecosystem changes caused by potash mining in Perm Krai for environmental risk assessment for new and existing potash enterprises.