High-salt Diet Research Articles

The Participation of Ferroptosis in Fibrosis of the Heart and Kidney Tissues in Dahl Salt-Sensitive Hypertensive Rats.

Salt-sensitive hypertension is often more prone to induce damage to target organs such as the heart and kidneys. Abundant recent studies have demonstrated a close association between ferroptosis and cardiovascular diseases. Therefore, we hypothesize that ferroptosis may be closely associated with organ damage in salt-sensitive hypertension. This study aimed to investigate whether ferroptosis is involved in the occurrence and development of myocardial fibrosis and renal fibrosis in salt-sensitive hypertensive rats. Ten 7-week-old male Dahl salt-sensitive (Dahl-SS) rats were adaptively fed for 1 week, then randomly divided into two groups and fed either a normal diet (0.3% NaCl, normal diet group) or a high-salt diet (8% NaCl, high-salt diet group) for 8 weeks. Blood pressure of the rats was observed, and analysis of the hearts and kidneys of Dahl-SS rats was conducted via hematoxylin-eosin (HE) staining, Masson staining, Prussian blue staining, transmission electron microscopy, tissue iron content detection, malondialdehyde content detection, immunofluorescence, and Western blot. Compared to the normal diet group, rats in the high-salt diet group had increases in systolic blood pressure and diastolic blood pressure (P < 0.05); collagen fiber accumulation was observed in the heart and kidney tissues (P < 0.01), accompanied by alterations in mitochondrial ultrastructure, reduced mitochondrial volume, and increased density of the mitochondrial double membrane. Additionally, there were significant increases in both iron content and malondialdehyde levels (P < 0.05). Immunofluorescence and Western blot results both indicated significant downregulation (P < 0.05) of xCT and GPX4 proteins associated with ferroptosis in the high-salt diet group. Ferroptosis is involved in the damage and fibrosis of the heart and kidney tissues in salt-sensitive hypertensive rats.

Drug pair of Cornus officinalis and Radix achyranthis bidentatae improves renal injury of hypertension by regulating metabolic reprogramming mediated by eNOS

ObjectiveTo explore the effects and possible mechanisms of the drug pair Cornus officinalis and Radix achyranthis bidentatae (SYR-NX) on improving hypertensive kidney damage. MethodSYR-NX, a formulation of Cornus officinalis and Radix Achyranthis Bidentatae with a dose ratio 1:2.5, was used in this experiment. We investigated the effects of SYR-NX on spontaneously hypertensive rats (SHR) fed with a high-salt diet and Human Kidney-2 (HK2) cells exposed to hypoxia. After 8 weeks of treatment with SYR-NX, blood pressure was tested, and β 2-Microglobulin(β2-MG), blood creatinine (S-cr), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH), M2 pyruvate kinase (PKM2), adenosine triphosphate (ATP), pyruvate, lactate, connective tissue growth factor (CTGF) and tumor necrosis factor-α (TNF-α)were measured. HK2 cells pre-treated with SYR-NX were cultured in a three-gas hypoxic incubator chamber (5 % CO2, 1 % O2, 94 % N2) for 12 h, and then eNOS, PKM2, NADPH, ATP, pyruvate, lactate, CTGF and TNF-α were assessed. ResultsSYR-NX significantly reduced SBP, DBP, β2-MG, S-cr, PKM2, pyruvate, lactate, CTGF and TNF-α, and increased eNOS, NADPH, and ATP. ConclusionSYR-NX can regulate metabolic reprogramming through eNOS and improves hypertensive kidney injury.

High salt diet alleviates disease severity in native experimental autoimmune uveitis.

Recent studies reported a link between high salt diet (HSD) and clinical exacerbation in mouse models of autoimmune diseases, mainly through the induction of pathogenic Th17 cells and/or HSD-induced dysbiosis. However, the topic remains controversial and not fully understood. In this study, we investigated the effects of HSD on the development of experimental autoimmune uveitis (EAU) in C57BL/6J mice. Unexpectedly, our data showed a significant attenuating effect of HSD on disease severity of native EAU, induced by direct immunization with IRBP peptide. That said, HSD had no effect on EAU disease severity induced by adoptive transfer of semi-purified auto-reactive IRBP-specific T lymphocytes. Accordingly, HSD did not affect IRBP-specific systemic afferent immune response as attested by no HSD-linked changes in T lymphocytes proliferation, cytokine production and Treg proportion. Gut microbiota analysis from cecal samples in naïve and EAU mice demonstrated that HSD affected differentially α-diversity between groups, whereas β-diversity was significantly modified in all groups. Unknown Tannerellaceae was the only taxon associated to HSD exposure in all treatment groups. Interestingly, a significantly higher abundance of unknown Gastranaerophilales, with potential anti-inflammatory properties, appeared in HSD-fed native EAU mice, only. In conclusion, our study suggests a possible impact of HSD on gut microbiota composition and consequently on development and clinical severity of EAU. Further studies are required to investigate the potential beneficial role of Gastranaerophilales in EAU.

Dietary Cinnamaldehyde Activation of TRPA1 Antagonizes High-Salt-Induced Hypertension Through Restoring Renal Tubular Mitochondrial Dysfunction.

The renal proximal tubule (RPT) plays a pivotal role in regulating sodium reabsorption and thus blood pressure (BP). Transient receptor potential ankyrin 1 (TRPA1) has been reported to protect against renal injury by modulating mitochondrial function. We hypothesize that the activation of TRPA1 by its agonist cinnamaldehyde may mitigate high-salt intake-induced hypertension by inhibiting urinary sodium reabsorption through restoration of renal tubular epithelial mitochondrial function. Trpa1-deficient (Trpa1-/-) mice and wild-type (WT) mice were fed standard laboratory chow [normal diet (ND) group, 0.4% salt], standard laboratory chow with 8% salt [high-salt diet (HS) group], or standard laboratory chow with 8% salt plus 0.015% cinnamaldehyde [high-salt plus cinnamaldehyde diet (HSC) group] for 6 months. Urinary sodium excretion, reactive oxygen species (ROS) production, mitochondrial function, and the expression of sodium hydrogen exchanger isoform 3 (NHE3) and Na+/K+-ATPase of RPTs were determined. Chronic dietary cinnamaldehyde supplementation reduced tail systolic BP and 24-hour ambulatory arterial pressure in HS-fed WT mice. Compared with the mice fed HS, cinnamaldehyde supplementation significantly increased urinary sodium excretion, inhibited excess ROS production, and alleviated mitochondrial dysfunction of RPTs in WT mice. However, these effects of cinnamaldehyde were absent in Trpa1-/- mice. Furthermore, chronic dietary cinnamaldehyde supplementation blunted HS-induced upregulation of NHE3 and Na+/K+-ATPase in WT mice but not Trpa1-/- mice. The present study demonstrated that chronic activation of Trpa1 attenuates HS-induced hypertension by inhibiting urinary sodium reabsorption through restoring renal tubular epithelial mitochondrial function. Renal TRPA1 may be a potential target for the management of excessive dietary salt intake-associated hypertension.

Antihypertensive activity of Lycopodium cernuum (Lycopodiaceae) hydro alcoholic extract on rats

The aim of this work was to study the antihypertensive activity of the hydroalcoholic extract of the aerial part of Lycpodium cernuum in rats made experimentally hypertensive with a hypersodic diet. Its hypotensive effect was studied in vivo and its vascular effect was studied in vitro on the isolated aorta. The normal blood pressure of the rats was 103.83 ± 3.7 / 82.13 ± 2 mmHg (systole/diastole). After 21 days on the high-salt diet, their blood pressure rose to 208 ± 2.2 / 150.06 ± 2.5 mmHg. This pressure returned to normal after 22 days in the control lot compared to 6, 10 and 16 days in the animals treated with the extract at 400, 200 and 100 mg/kg of the extract orally administered. In vitro, the extract relaxes the isolated aorta contracted with norepinephrine 10-3 M with an EC50 equal to 0.65 ± 0.02 mg/ml. These results show that Lycopodium cernuum extract has an antihypertensive activity in rats by its vasodilatory effect. This activity could be due to the presence of phenolic compounds and/or alkaloids in the extract.

Increased oxidative stress alters coronary microvascular tone in exercising swine with multiple comorbidities

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Cardiovascular Alliance Background/Objectives We have previously shown, in female swine that the combination of three cardiovascular risk factors (diabetes mellitus (DM), hypercholestolemia (HC) and chronic kidney disease (CKD)) induces systemic inflammation, oxidative stress, coronary endothelial dysfunction and impaired myocardial perfusion due to impaired nitric oxide (NO) bioavailability. In the present study we hypothesized that the reduced bioavailability of nitric oxide (NO) is the result of increased production of reactive oxygen species (ROS) and ROS scavenging will restore myocardial oxygen balance thereby improving myocardial perfusion. Methods 12 female swine, with DM (streptozotocin 3x50mg/kg iv), CKD (renal embolization), and HC (high fat, high sugar, high salt diet) and 9 female healthy swine on normal pig chow (Normal) were included. The effect of ROS scavenging on the coronary microvascular tone control was studied in vivo, at rest and during graded treadmill exercise before and after infusion of the ROS scavengers (MPG+Tempol). Additionally, vasodilation to bradykinin was studied in the presence of MPG and TEMPOL and after subsequent addition to catalase, in isolated left ventricle small coronary arteries, in vitro using wire-myography. Results 6 months of DM+HC+CKD resulted in hyperglycemia (18.0±1.3 vs 8.4±0.9mmol/L), hypercholesterolemia (13.4±2.1 vs 1.7±0.1mmol/l), renal dysfunction (plasma creatinine: 166±9 vs 119±3µmol/l), and systemic inflammation (TNFα: 63±8 vs 41±4pg/ml, all P&amp;lt;0.05) associated with impaired endothelium-dependent vasodilator response to bradykinin in vitro. Furthermore, myocardial oxidative stress was increased in DM+HC+CKD animals (8-isoprostane 13±1 vs 10±1pg/ml) and total antioxidant capacity was reduced (0.67±0.02 vs 0.72±0.01nmol Trolox eq./mg protein, both P&amp;lt;0.05). Surprisingly, in vivo ROS scavenging resulted in an even further increase in myocardial oxygen extraction in DM+HC+CKD while it had no effect in Normal, indicating the involvement of a vasodilator ROS (most likely H2O2) in the control of coronary microvascular tone. This was supported by increased activity of catalase in the myocardium of DM+HC+CKD animals (44±3 vs 31±4nmol/min/ml, both P&amp;lt;0.05) and the vascular relaxation response to bradykinin when catalase was added on top of the ROS scavenging in vitro. Conclusion In swine exposed for 6 months to multiple comorbidities, impaired myocardial perfusion is mediated by a loss of NO bioavailability due to increased oxidative stress which was partially compensated by increased H2O2-mediated coronary vasodilation.

Impaired cardiac mitochondrial bioenergetics contribute to loss of cardiac function, in a porcine multiple risk factor model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch CardioVascular Alliance (an initiative with support of the Dutch Heart Foundation) Grant 2020B008 RECONNEXT, and the German Center for Cardiovascular Research (DZHK; 81Z0600207) Background Heart failure with preserved ejection fraction lacks targeted therapies, due to insufficient understanding of pathogenesis. A multiple comorbidity swine model (MCS) was developed by exposure to three risk factors for six months including diabetes (streptozotocin), chronic kidney disease (renal embolization), and an unhealthy diet (high-fat, high salt diet). An additional risk factor ovariectomy was added on top of the MCS model (MCS+). MCS and MCS+ presented with left ventricular diastolic dysfunction, oxidative stress, coronary microvascular dysfunction and inflammation. Furthermore, myocardial oxygen consumption was increased given the same level of cardiac work (Fig. A), suggesting an impairment in myocardial mitochondrial function. Using a combined-omics approach, the purpose of this study is to unravel alterations in gene expression and proteome abundance regarding mitochondrial function in MCS swine. Methods 15 MCS, 4 MCS+ and 10 healthy control female swine were included in the study. Proteome analysis and single nuclei RNA sequencing, were performed on frozen MCS left ventricle myocardial samples. Cardiac mitochondrial function was measured by an O2k-FlouRespirometer ex vivo in MCS+ swine versus control. Results Proteome analysis showed a reduced abundance of proteins involved in branched-chain amino acid (BCAA) catabolism (Fig. B). Single nuclei RNA sequencing of a subgroup of the same animals demonstrated a downregulation of these genes specifically in cardiomyocyte subpopulations. BCAA can act as a source of ATP via oxidative phosphorylation and can modulate mitochondrial substrate utilisation, and impair mitochondrial function, resulting in increased mitochondrial production of reactive oxygen species (ROS). Mitochondrial function was measured in fresh myocardial tissue, using pyruvate/malate/glutamate as substrate. Low mitochondrial respiratory sensitivity to ADP was observed 26±2.5 in MCS+ vs 43.6±1.5 pmol/(s*mg tissue) in healthy control, reflecting low oxidative capacity. Moreover, the respiratory rate after uncoupling by FCCP was reduced in MCS+ versus healthy control (73.6±11.7 vs 118.4±15.6 pmol/(s*mg tissue). These findings are consistent with our data in the MCS swine and indicate impaired BCAA in the myocardium of animals with comorbidities, which is associated with increased ROS levels (8-isoprostane 12.9±0.8 pg/mg protein in MCS vs 10.3±0.5 in healthy animals) and impaired myocardial efficiency during exercise (Fig. A). Conclusion A combined omics approach suggested an alteration in cardiac mitochondria. Respiratory analyses showed impaired mitochondrial bioenergetics and increased oxidative stress, which can contribute to diastolic dysfunction.

The impact of high-salt diet on asthma in humans and mice: Effect on specific T-cell signatures and microbiome.

The rise in asthma has been linked to different environmental and lifestyle factors including dietary habits. Whether dietary salt contributes to asthma incidence, remains controversial. We aimed to investigate the impact of higher salt intake on asthma incidence in humans and to evaluate underlying mechanisms using mouse models. Epidemiological research was conducted using the UK Biobank Resource. Data were obtained from 42,976 participants with a history of allergies. 24-h sodium excretion was estimated from spot urine, and its association with asthma incidence was assessed by Cox regression, adjusting for relevant covariates. For mechanistic studies, a mouse model of mite-induced allergic airway inflammation (AAI) fed with high-salt diet (HSD) or normal-salt chow was used to characterize disease development. The microbiome of lung and feces (as proxy for gut) was analyzed via 16S rRNA gene based metabarcoding approach. In humans, urinary sodium excretion was directly associated with asthma incidence among females but not among males. HSD-fed female mice displayed an aggravated AAI characterized by increased levels of total IgE, a TH2-TH17-biased inflammatory cell infiltration accompanied by upregulation of osmosensitive stress genes. HSD induced distinct changes in serum short chain fatty acids and in both gut and lung microbiome, with a lower Bacteroidetes to Firmicutes ratio and decreased Lactobacillus relative abundance in the gut, and enriched members of Gammaproteobacteria in the lung. High dietary salt consumption correlates with asthma incidence in female adults with a history of allergies. Female mice revealed HSD-induced T-cell lung profiles accompanied by alterations of gut and lung microbiome.

Chronic High-Salt Diet Activates Tumor-Initiating Stem Cells Leading to Breast Cancer Proliferation.

Several chronic inflammatory diseases have been linked to high-salt (HS) diets. Chronic inflammation is an established causative hallmark of cancer. However, a direct role of HS diets in tumorigenesis is yet to be defined. Previous orthotopic murine breast tumor studies have shown that short-term HS diets caused inhibition of tumor growth through the activation of cytotoxic adaptive immune responses. However, there have been experimental challenges in developing a viable chronic HS-diet-based murine tumor model. To address this, we have developed a novel chronic HS diet tumor model through the sequential passaging of tumor cells in mice under HS dietary conditions. Two orthotopic murine triple-negative breast cancer models, 4T1 tumor cells injected into BALB/c mice and Py230 tumor cells injected into C57Bl/6 mice, were utilized in our study. For the HS diet cohort, prior to orthotopic injection with tumor cells, the mice were kept on a 4% NaCl diet for 2 weeks. For the regular salt (RS) diet cohort, the mice were kept on a 1% NaCl diet. Following syngeneic cancer cell injection, tumors were allowed to grow for 28 days, following which they were collected to isolate immune cell-depleted cancer cells (passage 1, P1). The tumor cells from P1 were reinjected into the next set of non-tumor-bearing mice. This procedure was repeated for three cycles (P2-P4). In P1, compared to the RS diet cohort, we observed reduced tumor kinetics in both murine tumor models on the HS diet. In contrast, by P4, there was significantly higher tumor progression in the HS diet cohort over the RS diet cohort. Flow cytometry analysis demonstrated an 8-fold increase in tumor-initiating stem cells (TISCs) from P1 to P4 of the HS diet cohort, while there were no significant change in TISC frequency with sequential passaging in the RS diet cohort. Molecular studies showed enhanced expression of TGFβR2 and CD80 on TISCs isolated from the P4 HS diet cohort. In vitro studies demonstrated that TGFβ stimulation of these TISCs increased the cellular expression of CD80 molecules. Further, the chronic HS diet selectively induced the glycolytic metabolic phenotype over the mitochondrial oxidative phosphorylation phenotype in TISCs, which is needed for the production of metabolites during tumor cell differentiation and proliferation. The infiltrating CD8 and CD4 T-lymphocytes in P4 tumors demonstrated increased expression of the immune checkpoint inhibitor (ICI) CTLA4, a known binding partner of CD80, to cause immune exhaustion and pro-tumorigenic effects. Interestingly, anti-TGFβ monoclonal antibodies (mAbs) played a synergistic role in further enhancing the anti-tumor effect of anti-CTLA4 mAb. In summary, our findings demonstrated that chronic HS diet increased the frequency of TISCs in tumors leading to blunting of cytotoxic adaptive immune responses causing tumor proliferation. Furthermore, a combination of anti-TGFβ with current ICI-based immunotherapies could exert more favorable anti-cancer clinical outcomes.

#2906 Kidney immune cells and endothelial cells can be modulated by low-salt and high-salt diets

Abstract Background and Aims Kidney immune cells and endothelial cells are involved in pathogenesis of kidney ischemia-reperfusion injury and potential therapeutic targets to improve kidney transplant outcomes. We hypothesized that dietary salt intake can change kidney immune cells and endothelial cells. Method C57BL/6 mice were treated with normal diet, low-salt diet, or high-salt diet. After 6 weeks of allocated diets, mice were euthanized, and their kidneys were collected. Leukocyte infiltration was assessed with CD45 immunohistochemistry. Lymphocytes were isolated from kidneys and were studied by flow cytometry. Human kidney transplant tissues obtained by a protocol biopsy at 2 weeks after kidney transplantation were stained with CD45, CD3, CD20, and CD31. Expression of each marker was quantified using an automated imaging analysis system and analyzed per donors’ pre-donation 24 h urine sodium levels. Induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from healthy individuals and end-stage kidney disease (ESKD) patients were treated with different sodium concentrations, and their proliferation was assessed. Results Kidneys from mice fed with high-salt diet had higher kidney leukocytes (CD45+) than those fed with normal diet (1.9 ± 0.3% vs 0.9 ± 0.1%, P = .043). Effector-memory CD4+ T cells (normal 44.2 ± 2.6%; low-salt 57.1 ± 3.5%, P = .020; high-salt 62.1 ± 2.5% of CD4+ T cells, P = .002) and mature B cells (70.0 ± 1.5%; 79.1 ± 1.4%, P = .001; 76.7 ± 1.6% of CD19+ cells, P = .015) increased by low-salt and high-salt diets, whereas naïve CD4+ T cells deceased (45.9 ± 2.1%; 32.7 ± 3.2%, P = .016; 27.8 ± 3.1% of CD4+ T cells, P = .001) in kidneys. Human kidney transplants from donors under low-salt diet showed lower total leukocytes (CD45+, 0.3 ± 0.1% vs 0.9 ± 0.3%, P = .019) with lower T and B cells (CD3+, 1.8 ± 0.2% vs 3.5 ± 0.4%, P&amp;lt;.001; CD20+, 0.8 ± 0.1% vs 2.1 ± 0.6%, P = .011) and higher capillary density (CD31+, 29.0 ± 2.2% vs 16.1 ± 1.8%, P = .009) compared to those from donors under high-salt diet. The proliferation of iPSC-ECs from healthy individuals and ESKD patients after hypoxia was reduced under higher sodium concentrations. Conclusion High-salt and low-salt diets affect kidney immune cell compositions and numbers as well as endothelial cells. Reconstitution of kidney microenvironment by modifying dietary salt intake could be a novel approach to improve kidney transplant outcomes.

#1755 Cardio-protective effect of soy proteins during long-term consumption of Cynomolgus Monkeys high-saline diet

Abstract Background and Aims High salt intake is accompanied by the development of cardiovascular disorders, not always associated with an increase in blood pressure (BP). Recently, it has been found that the introduction of soy protein into the diet can have some nephroprotective and cardioprotective effects in chronic kidney disease. Considering the cardioprotective effect of soy proteins in chronic kidney disease, the question arose about the possibility of using soy proteins to neutralize the damaging effect of a high-salt diet on the cardiovascular system. This work aims to study the effect of long-term use of a diet high in NaCl and soy protein on myocardial remodeling in monkeys. Method The study was performed on male Macaca fascicularis. The animals were divided into three groups (6 animals each), comparable in weight and age (6-8 years, body weight - 5.15-9.7 kg). The control group received a standard diet (2 g NaCl/kg feed). The second was a high salt diet (8 g NaCl/kg feed), and the third was a high salt diet and SUPRO760 soy protein (200 g/kg feed). Echocardiography (EchoCG), registration of BP and heart rate were performed at baseline, after 4 and 12 months. EchoCG was performed with a sector transducer with a frequency of 3–5 MHz on the Chison SonoTouch 60 ultrasound system (China). EchoCG was performed in B_mode (two-dimensional scanning), M_mode (one-dimensional scanning), as well as in pulse and tissue Doppler modes. Statistical analysis was performed using parametric statistics: mixed ANOVA in comparing data from 3 study groups (with post hoc analysis using Tukey criterion) and single factor variance analysis for related samples in studying the dynamics of one or another index in each group during the 12-month follow-up. Differences were considered significant at p &amp;lt; 0.05. Results In animals of all three groups for 12 months, blood pressure (BPsis-109±10.09, BPdias – 60 ± 7.42 mm Hg) and heart rate (77 ± 12.61 min-1) did not change significantly (p&amp;gt;0. 05). However, in macaques on a high-salt diet, deterioration in left ventricular (LV) systolic and diastolic function was noted by the end of the study. Macaques of this group had a decrease in the LV shortening fraction (27.9±2.69%) and an increase in the thickness of the posterior wall of the LV in systole (0.69±0.039 cm), LV mass (19.60±2.83 g), compared with animals additionally receiving soy proteins (34.9±6.57%; 0.57 0.0559 cm; 14.86±2.94 g, respectively; all p &amp;lt; 0.05). In addition, the monkeys receiving soy isolate did not differ from the control in these parameters. By the time the study was completed, the animals in this group had significantly increased the magnitude of systolic excursion of the mitral annulus plane, and signs of improvement in LV diastolic function appeared in the form of an increase in the maximum rate of excursion of the mitral valve fibrous annulus in early diastole, as well as a decrease in the E/e' ratio. The thickness of the interventricular septum also decreased (0.42±0.04 cm; in the high-salt group - 0.51±0.08 cm), which can be interpreted as a sign of positive myocardial remodeling. Conclusion Thus, high salt intake leads to adverse structural and functional disorders of the heart in cynomolgus monkeys, not associated with an increase in blood pressure. The inclusion of soy isolate in the diet reduces the negative effects of a high-salt diet on the cardiovascular system.

#1463 Assessment of salt-sensitive hypertension in a novel Lanosterol Synthase knock-in mouse model

Abstract Background and Aims The diversity in blood pressure (BP) response to various salt intakes, known as Salt Sensitivity (SS), exhibits significant differences among individuals within the hypertensive subjects relative to the general population. Hypertension (HT) and SS are correlated to elevated levels of plasma Endogenous Ouabain (EO). We aimed at delineating the role of the missense variation rs2254524 (Val642Leu; C&amp;gt;A) within the Lanosterol Synthase gene (LSS), encoding for an enzyme involved in steroid biosynthesis. Individuals with the AA LSS variant, adherent to a low-salt diet, had a more significant BP reduction compared to those with CC genotype. Method Utilising CRISPR-Cas9, we established a knock-in mouse model carrying the analogous LssV643L/V643L. Male mice were subjected to a normal-salt diet (0.5% NaCl), high-salt diet (4% NaCl), or low-salt diet (&amp;lt;0.03% Na) and BP was monitored every two days using a tail-cuff system for 10 days. Blood and urine were collected separately in metabolic cages. Results Homozygous mice for Lss mutation demonstrated normal viability and healthy phenotype, undistinguishable from the WT. At baseline, the Lss AA mice were affected by a significant augmentation in kidney weight at 3 and 12 months of age, relative to WT. The Lss V643L does not influence EO levels or systolic BP (SBP), specifically at 3 and 12 months, but affects the SBP response to dietary salt. AA mice showed a high SBP in a high-salt diet against control diet at the same time intervals. Moreover, the Lss variant impacted the pressure-natriuresis relationship, with the mutated AA strain showing a less steep curve both in control diet versus the high-salt diet and in control diet versus the low-salt diet, displaying the SS phenotype of the AA mice. The 12-month-old mice in high-salt diet displayed cardiac hypertrophy with the AA genotype exhibiting an elevated prevalence of cardiac fibrosis. The level of Lss mRNA decreased in the adrenal glands in response to a high-salt diet. RNASeq analysis identified distinct patterns in several Slc genes, Cbr2 and Arhgap26 within the kidneys of these mice, in control or high-salt dietary conditions. Conclusion The established LssV643L/V643L mouse model effectively mirrors the SS-HT phenotype. Our findings highlight the significance of the Lss in modulating BP in response to salt stimuli suggesting a potential organ damage pathway requiring further exploration in older mice.

Augmentation of Nitric Oxide Deficient Hypertension by High Salt Diet Is Associated With Reduced TNF-α Receptor Type 1 Expression in the Kidneys.

High salt (HS) intake induces an augmented hypertensive response to nitric oxide (NO) inhibition, though it causes minimal changes in blood pressure (BP) in NO intact condition. The cause of such augmentation is not known. HS induces tumor necrosis factor-alpha (TNFα) production that causes natriuresis via activation of its receptor type 1 (TNFR1). We hypothesized that NO deficiency reduces renal TNFR1 activity, leading to enhanced sodium retention and hypertension. We examined the changes in renal TNFR1 protein expression (Immunohistochemistry analyses) after HS (4% NaCl) intake in wild-type mice (WT, C57BL6) treated with a NO synthase (NOS) inhibitor, nitro-l-arginine methyl ester (L-NAME; 0.05 mg/min/g; osmotic mini-pump), as well as in endothelial NOS knockout mice (eNOSKO) and compared the responses in WT mice with normal salt (NS; 0.3% NaCl) intake. BP was measured with tail-cuff plethysmography and 24-hour urine collections were made using metabolic cages. HS alone did not alter mean BP in untreated mice (76 ± 3 to 77 ± 1 mm Hg) but induced an augmented response in L-NAME treated (106 ± 1 vs. 97 ± 2 mm Hg) and in eNOSKO (107 ± 2 vs. 89 ± 3 mm Hg) mice. The percentage area of TNFR1 expression in renal tissue was higher in WT + HS (4.1 + 0.5%) than in WT + NS mice (2.7 ± 0.6%). However, TNFR1 expression was significantly lower in L-NAME treated WT + NS (0.9 ± 0.1%) and in eNOSKO + NS (1.4 ± 0.2%) than in both WT + NS and WT + HS mice. These data indicate that TNFR1 activity is downregulated in NO deficient conditions, which facilitates salt retention leading to augmented hypertension during HS intake.

#1749 Influence of high saline diet on myocardial remodeling, microRNA-21i and NFkB expression in myocardium

Abstract Background and Aims There is now increased interest in the study of morpho-functional myocardial changes in excess NaCl consumption, which develop independently of AD dynamics. Myocardial remodeling may involve microRNAs that modulate gene expression at the post-transcription level. However, their role in this process has not been fully explored. The purpose of the study is to assess the levels of expression of the nucleic transcription factor kV (NFkB), microRNA-21 and structural changes in the myocardium at long-term consumption of Wistar diet with high (8%) table salt content. Method Male Wistar rats (body mass 280.5 42.7 g) were divided into two groups of 10 animals. For 4 months, the control rats (NaCl) received a standard diet (0.34% NaCl), and the animals of the other group received a high salt diet (8% NaCl) (HS). Laboratory food varied only in NaCl content. 4 months later in rats systolic AD was measured, myocardial mass index (IMM) was evaluated, myocardial histology was performed (paraffin coloration - hematoxylin-eosin, Masson), relative expression levels of my RNA-determined 21 and NFkB in the myocardium, sodium content in the urine and blood serum. Statistical analysis used the t-Student criterion and the Mann-Whitney test. The differences were considered significant at p &amp;lt; 0.05. Results Excess NaCl (8%) in the diet had no effect on animal body weight (370.5 32.0 g in HS group, 393.0 21.4 g in NS group, p &amp;gt; 0.05) and systolic AD. In the HS-Group, the average AD was 134.5 8.9 mm old, and in the NS-Group - 134.8 5.2 mm old. (p &amp;gt; 0.05). However, HS-group IMM rats were 25% higher than NS-group rats (p &amp;lt; 0.05). The daily water consumption in the HS group was on average 33% higher than in the control, the daily diuresis also increased (10.5 5.7 ml against 5.7 2.9 ml in the NS-group, p &amp;lt; 0.001) and the sodium content in the urine (81.25.72 mmol/l against 161.18.57 mmol in the NS-01). There was no difference in the sodium content of the blood serum. Hypertrophy of cardiomyocytes, protein dystrophy, and increased wall thickness of arterial vessels (NS-20,42 3,53, HS-27,61 4,93, µm were identified in the HS-group; p &amp;lt; 0.01). The area of perivascular fibrosis in rats of the HS group (14876 ± 3803.7 µm2) was almost 1.8 times higher than that in the NS group (8426.0 ± 3012.2 µm2, p &amp;lt; 0.03). The thickness of cardiomyocytes (NS-12.97 1.53, HS-20.28 1.90 µm, p &amp;lt; 0.001) also increased in rats on the HS diet. In animals of the HS group, the relative expression levels of NFkB (more than 2 times) and microRNA-21 (almost 6 times) in the myocardium increased compared to the NS group. It can be assumed that the adverse effect on the cardiovascular system of high-salt diets is partially realized through NFκB-associated signaling pathways and the activation of microRNA-21. Conclusion Long-term use of a high-salt diet in Wistar rats results in myocardial remodeling unrelated to changes in AD levels. In this case, the high salt intake effects on the myocardium may be mediated by an increase in NFkBy and microRNA-21 expression levels.

#1138 Urine proteomic characterisation of DahlS rat model shows robust translatability of molecular profile with human hypertensive kidney damage

Abstract Background and Aims The role of hypertension in CKD is complex. Hypertensive nephropathy or arterionephrosclerosis is caused by prolonged periods of poorly controlled high blood pressure and is characterized by gradual loss of kidney function with various grades of proteinuria over a period of many years. This complexity requires novel analysis strategies for a successful understanding of pathomechanisms, drug target and biomarker identification including well characterized translatable models where the DalhS rats could be relevant. Method We analysed the urinary proteome of hypertensive Dahl S rats at baseline and 2, 4, and 7-weeks of high salt diet (4%, n = 5 in each cohort). A TMT MS/LC method was performed to obtain proteomics profile of the urine, to then filter, normalize and apply a differential analysis based on linear models and empirical Bayes statistics (limma) in R language. Additionally, we did a pathway enrichment analysis using IPA. Finally, to assess the translatability of the findings, we compared the rat urinary proteome data with publicly available urinary proteome from patients with CKD. Results The DalhS rats developed hypertension, proteinuria, and glomerulosclerosis. In the urine, we identified a total of 36 differentially expressed proteins comparing 4 weeks of high salt diet (HSD) versus the baseline, and 85 differentially expressed proteins at 7 weeks of HSD. Pathway analysis of the differentially abundant urinary proteome showed robust enrichment of pathways related to hypertensive kidney damage including ‘acute phase response signaling’, a pathway that activates rapidly in inflammatory settings and can sustain chronically, and liver / retinoid-x-receptor (LXR/RXR) activation​, which was also activated. Additionally, the coagulation system and complement cascade also showed positive z-scores in high-salt-exposed rats (after 7 weeks). Moreover, we observed an inactivation of several immune related pathways such as IL-12 signaling and STAT3 pathways. Finally, analysis of publicly available data of the urinary proteome of CKD patients (PMID 34963468, N = 40) exhibited enrichment of more than 20 pathways in common with the DalhS cohort, suggesting a high translatability between the urinary proteome of the animal model and patients. Specifically, we observe activation of acute phase response signalling, LXR/RXR activation, and complement cascade in DalhS and human datasets, as well as inhibition of IL-12 and STAT3 signaling pathways. Conclusion Molecules targeting the complement pathway are under intensive development. Prior development of LXR agonists have been limited by Ph1 findings. The findings with acute-phase response, IL-12 and STAT3 pathways suggests the importance of immune-driven damage mechanisms in hypertensive CKD. Identified proteins and enriched pathways in this work provide a rich resource for future investigation. In conclusion, the Dahl S rat model urinary proteome captures multiple underlying mechanisms of the human hypertensive CKD phenotype.

#1682 Myocardial remodeling in Wistar rats with renal dysfunction treated with a high-salt ration

Abstract Background and Aims High food intake of NaCl is the traditional factor of increase in blood pressure (BP) and cardiovascular risks also hurts kidneys. The purpose of the paper is to evaluate the morphological changes in the myocardium of normotensive Wistar rats with an early stage of renal dysfunction treated with a diet with increased concentration of salt for a long time. Method 30 Wistar rats (age 2.5-3 months) were studied and divided into three groups of 10 each. In the first, control, sham-operated (SO) rats, receiving standard diet (0.34% NaCl), in the second, rats subjected to resection of ¾ of the kidney parenchyma, receiving a standard diet (NE group), in the third, rats subjected to ¾ NE and those receiving a high-sodium diet (4% NaCl, high-salt diet, HSD). The feeds differed only in the %NaCl content. The ¾ NE model was created in two stages with an interval of 7 days. 20 mg/kg Zoletil 100 and 0.05 ml of Xylazine were used as anesthesia. At the first stage, the poles of the left kidney (1/2 part of the kidney) were removed, at the second stage, the right kidney was completely removed. The duration of the experiment was 4 months. At the end of the experiment, systolic BP at the tail was measured in awake rats using the cuff method (ELEMA electromanometer, Sweden). After removal from the experiment, blood was collected from the rats for biochemical analysis, and the LVMI was calculated as the ratio of LV mass to the body weight of the animal (mg/g). Myocardial fragments were placed in formalin, and sections 1.5–2 µm thick were prepared from paraffin blocks. The preparations were stained with Hematoxylin and Eosin and Masson's trichrome and used for histological analysis. The statistical significance of intergroup differences was determined using the Mann–Whitney test and Student's t test. Results During the observation period, BP in the SO group of animals did not change: initially - 120 [120;125] mm Hg, after 4 months - 120 [120;125] mmHg, p = 0.527. In the group with NE on a standard diet, an increase in BP was noted - from 120 [120; 125] mmHg up to 135.0 [132.5;137.5] mmHg, p = 0.011. In the group with NE + HSD, the increase in BP levels was more pronounced - up to 140[140;148] mmHg, (p = 0.001, relative to the SO group). The serum urea level in the SO group was 4.7 [4.5;5.4] mmol/l, creatinine −32.5 [29.0;36.0] µmol/l, in the NE group - 10.0 [9.1;10.9] mmol/l, (p = 0.0001, relative to the SO group) and 69.0[64.0;74.0] µmol/l, (p = 0.0001), in the NE+ HSD group – 10.7 [9.8;11.0] mmol/l, (p = 0.0001) and 64.5[46.3;69.5] µmol/l, (p = 0.001), respectively. Thus, the groups of rats with early-stage kidney dysfunction, although they had higher levels of urea and creatinine in the blood serum relative to the SO group, did not differ significantly from each other in these indicators. LVMI increased in rats with NE on a standard diet (LR −2.07±0.05 mg/g, NE −2.14 ± 0.06 mg/g, p = 0.1591). Consumption of a high-salt diet aggravated the growth of myocardial mass in rats with NE (NE + HSD - 2.41 ± 0.14 mg/g, p = 0.0084 and p = 0.0372, respectively). At the histological level, the increase in LV myocardial mass in rats with NE was characterized by hypertrophy of cardiomyocytes and the formation of interstitial fibrosis. The changes were more pronounced in the group with NE + HSD. The diameter of cardiomyocytes in rats of the SO group was 12.9 ± 0.4 µm, in animals with NE on standard diet - 14.5 ± 0.6 µm, in rats with NE + HSD - 15.5 ± 1.6 µm (p&amp;lt;0 .01 and p &amp;lt; 0.02, relative to the SO group). The area of interstitial fibrosis also increased in rats with NE (10.5 ± 2.6%, in the SO group - 0.6 ± 0.2%, p = 0.0000) and was slightly higher in the NE + HSD group (13.6 ± 2.8%, p = 0.0000, relative to the SO group). In rats with NE, both on a standard diet and with HSD, at this stage of the formation of kidney pathology, the thickness of the nutrimyocardial arteries did not differ significantly from the SO indicator rats. Conclusion Long-term salt intake with increased salt addition in Wistar rats in the initial stages of renal dysfunction does not affect the level of urea and creatinine in the blood serum, but promotes an increase in BP and myocardial remodeling, constantly progressing in the progression of cardiomyocyte hypertrophy and interstitial fibrosis.

#2704 Effects of high dietary salt on immunome and microbiome composition – results from a randomized clinical trial

Abstract Background and Aims Increased dietary salt intake ranks among the most prominent nutritional risk factors worldwide and has been tied to arterial hypertension and all-cause mortality. The mechanisms responsible are incompletely understood and have recently been extended to include inflammatory and microbiome-associated mechanisms. Building on our preliminary data, the present study investigates whether a moderate increase in salt intake in healthy subjects primes host physiology to a more transiently unstable state, potentially leading to changes in the microbiome-immune axis. Method We conducted a prospective, randomized, double-blinded trial to evaluate the effect of high dietary salt in healthy participants (NCT03024567), where the intervention group (n = 19) was given 6 g of salt (NaCl capsules) in addition to the habitual salt intake, resulting in an average doubling of the salt intake recommended by expert societies. A placebo group (n = 19) received gelatin capsules for 14 days. Clinical parameters, fecal samples and PBMC were collected at baseline and day 14. Shotgun metagenomic sequencing, metabolomics (stool and serum), and single-cell sequencing (Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq)) of whole PBMCs and CD4+ sorted T cells (∼150 k single cells) was performed. Results Microbiome analysis confirms previous findings showing an increased dissimilarity of microbiome composition (Jensen-Shannon Divergence) under a high-salt. Differential abundance of the microbiome's functional space, as quantified by functional gut-specific modules, picked up a salt-specific module (M00494, NatK-NatR (sodium extrusion) two-component regulatory system) in the salt intervention group. CITEseq revealed a significant reduction of circulating NK cells and an increase in classical monocytes as well as changes to several T cell subsets. Furthermore, within several immune cell subsets, we detected differently expressed genes (DEGs) solely in the salt group. We found 602 DEG in naïve conventional T cells (Tconv) and 202 DEGs in non-naïve Tconv, e.g. belonging to NF-kB and T cell receptor signaling pathways. Conclusion Our placebo-controlled study is the first to utilize different omics techniques to investigate and identify high-salt-induced changes in the microbiome and immunome in healthy individuals that may be relevant for health-to-disease transition.

#417 Targeting parietal epithelial cell activation with mineralocorticoid receptor antagonism controls FSGS and crescentic glomerulonephritis

Abstract Background and Aims We have recently demonstrated that targeting specific pathways in parietal epithelial cells (PEC) can markedly alleviate experimental extracapillary glomerular injury [1]. Accumulating evidence has indicated the potential contributions of the mineralocorticoid receptor (MR) to the pathophysiology of chronic kidney disease. MR is expressed in endothelial cells, glomerular mesangial cells, podocytes, and distal tubular cells. Previous studies have shown that administering mineralocorticoid receptor antagonists has beneficial effects in various renal injury animal models, the role of the MR in extracapillary glomerulopathies is still elusive and mechanistically unclear. Methods To investigate the cell-specific role of the MR in PEC in the course of focal segmental glomerulosclerosis (FSGS) and crescentic glomerulonephritis (GN), we generated chimeric mice specifically lacking MR (Pec Cre Nr3c2lox/lox) in PECs using an inducible Cre recombinase system. Maladaptive FSGS was induced by combining a high-salt diet and deoxycorticosterone acetate (DOCA) with unilateral nephrectomy (DOCA-UniN). Crescentic glomerulonephritis (GN) was induced using the anti-glomerular basement membrane nephrotoxic serum (NTS) model. Results At baseline, Pec Cre Nr3c2wt/wt and Pec Cre Nr3c2lox/lox mice displayed no kidney morphology and function differences. When challenged by DOCA-UniN, PEC-selective Nr3c2 targeting significantly inhibited the expression of PEC activation markers Ki67, CD44, and FSGS lesions. Importantly, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function compared to Pec Cre Nr3c2 wt/wt counterparts. Likewise, Pec Cre Nr3c2lox/lox mice showed less albuminuria and preserved renal function in the NTS-induced CGN model. Crescents were also more numerous and organised in Pec Cre Nr3c2wt/wt mice than in Pec Cre Nr3c2lox/lox animals. Next, we examined whether pharmacological MR inhibition could alleviate the severity of crescentic GN. When Pec Cre Nr3c2wt/wt mice were orally given eplerenone for 14 days after the onset of the crescentic GN, they were significantly protected from renal injury and failure (decreased proteinuria, normal BUN and reduced number of crescent). Such global action was associated with less expression of the CD44 activation marker on PECs. Thus, genetic disruption of MR in PEC and pharmacological inhibition using eplerenone reduced glomerular expression of CD44 and crescent formation. Furthermore, kidney biopsies of individuals diagnosed with crescentic glomerulonephritis displayed increased expression of MR in PEC within FSGS and crescents. We next evaluated PEC migration under various conditions (DMSO, HB-EGF, Eplerenone, or HB-EGF+Eplerenone) for 24 hours. Eplerenone fully abrogated HB-EGF-induced PEC migration and Pcna, Snai1, and Vim mRNA expressions. Conclusion Altogether, these results indicate the critical role of MR in PEC activation during crescentic glomerulonephritis, along with the recently discovered CD9/EGFR/PDGFR [1] and CLDN1 pathways. This further supports the idea that the PEC phenotype switch is not a bystander event but plays a targetable critical active pathogenic role in FGSS and, more surprisingly, in crescentic GN. MR modulation using eplerenone may be a new therapeutic option for the management of such severe disease.

High salt diet induces cognitive impairment and is linked to the activation of IGF1R/mTOR/p70S6K signaling.

A high-salt diet (HSD) has been associated with various health issues, including hypertension and cardiovascular diseases. However, recent studies have revealed a potential link between high salt intake and cognitive impairment. This study aims to investigate the effects of high salt intake on autophagy, tau protein hyperphosphorylation, and synaptic function and their potential associations with cognitive impairment. To explore these mechanisms, 8-month-old male C57BL/6 mice were fed either a normal diet (0.4% NaCl) or an HSD (8% NaCl) for 3 months, and Neuro-2a cells were incubated with normal medium or NaCl medium (80 mM). Behavioral tests revealed learning and memory deficits in mice fed the HSD. We further discovered that the HSD decreased autophagy, as indicated by diminished levels of the autophagy-associated proteins Beclin-1 and LC3, along with an elevated p62 protein level. HSD feeding significantly decreased insulin-like growth factor-1 receptor (IGF1R) expression in the brain of C57BL/6 mice and activated mechanistic target of rapamycin (mTOR) signaling. In addition, the HSD reduced synaptophysin and postsynaptic density protein 95 (PSD95) expression in the hippocampus and caused synaptic loss in mice. We also found amyloid β accumulation and hyperphosphorylation of tau protein at different loci both in vivo and in vitro. Overall, this study highlights the clinical significance of understanding the impact of an HSD on cognitive function. By targeting the IGF1R/mTOR/p70S6K pathway or promoting autophagy, it may be possible to mitigate the negative effects of high salt intake on cognitive function.

High-Salt Diet Inhibits the Expression of Bmal1 and Promotes Atrial Fibrosis and Vulnerability to Atrial Fibrillation in Dahl Salt-Sensitive Rats.

Hypertension is a risk factor for atrial fibrillation (AF), and brain and muscle arnt-like protein 1 (Bmal1) regulate circadian blood pressure and is implicated in several fibrotic disorders. Our hypothesis that Bmal1 inhibits atrial fibrosis and susceptibility to AF in salt-sensitive hypertension (SSHT) and our study provides a new target for the pathogenesis of AF induced by hypertension. The study involved 7-week-old male Dahl salt-sensitive that were fed either a high-salt diet (8% NaCl; DSH group) or a normal diet (0.3% NaCl; DSN group). An experimental model was used to measure systolic blood pressure (SBP), left atrial ejection fraction (LAEF), left atrial end-volume index (LAEVI), left atrial index (LAFI), AF inducibility, AF duration, and atrial fibrosis pathological examination and the expression of Baml1 and fibrosis-related proteins (TNF-α and α-SMA) in left atrial tissue. DSH increased TNF-α and α-SMA expression in atrial tissue, level of SBP and LAESVI, atrial fibrosis, AF induction rate, and AF duration, and decreased Bmal1 expression in atrial tissue, the circadian rhythm of hypertension, and level of LAEF and LAFI. Our results also showed that the degree of atrial fibrosis was negatively correlated with Bmal1 expression, but positively correlated with the expression of TNF-α and α-SMA. We demonstrated that a high-salt diet leads to circadian changes in hypertension due to a reduction of Bmal1 expression, which plays a crucial role in atrial fibrosis and increased susceptibility to AF in SSHT rats.