Male Dahl Salt-sensitive Rats Research Articles

The Impact of Hydrogen Sulfide in the Paraventricular Nucleus on the MAPK Pathway in High Salt-Induced Hypertension.

The hypothalamic paraventricular nucleus (PVN) plays a central role in regulating cardiovascular activity and blood pressure. We administered hydroxylamine hydrochloride (HA), a cystathionine-β-synthase inhibitor, into the PVN to suppress endogenous hydrogen sulfide and investigate its effects on the mitogen-activated protein kinase (MAPK) pathway in high salt (HS)-induced hypertension. We randomly divided 40 male Dahl salt-sensitive rats into 4 groups: the normal salt (NS) + PVN vehicle group, the NS + PVN HA group, the HS + PVN vehicle group, and the HS + PVN HA group, with 10 rats in each group. The rats in the NS groups were fed a NS diet containing 0.3% NaCl, while the HS groups were fed a HS diet containing 8% NaCl. The mean arterial pressure was calculated after noninvasive measurement using an automatic sphygmomanometer to occlude the tail cuff once a week. HA or vehicle was infused into the bilateral PVN using Alzet osmotic mini pumps for 6 weeks after the hypertension model was successfully established. We measured the levels of H 2 S in the PVN and plasma norepinephrine using enzyme linked immunosorbent assay. In addition, we assessed the parameters of the MAPK pathway, inflammation, and oxidative stress through western blotting, immunohistochemical analysis, or real-time polymerase chain reaction. In this study, we discovered that decreased levels of endogenous hydrogen sulfide in the PVN contributed to the onset of HS-induced hypertension. This was linked to the activation of the MAPK signaling pathway, proinflammatory cytokines, and oxidative stress in the PVN, as well as the activation of the sympathetic nervous system.

Abstract P169: SGLT2 Colocalizes on the Nucleus Tractus Solitarii Neurons in Normotensive and Hypertensive Rats in a Location-Dependent Manner

Introduction/Background: Hypertension affects ~45% adults in the US and is associated with a high sodium diet. The nucleus tractus solitarii (nTS) in the brainstem influences blood pressure through the interplay between glutamate and GABA. Sodium-glucose cotransporter 2 (SGLT2) is a protein recently found within the nTS, and its expression in the nTS may impact blood pressure. In this study, we investigated the distribution of SGLT2 in the nTS of normotensive and hypertensive rats, focusing on its presence in glutamatergic (Glu) and GABAergic neurons. Within the nTS, increases in glutamate lowers blood pressure, and increases of GABA raises blood pressure. The interaction between SGLT2 and glutamate/GABAergic neurons within the nTS is not well-understood. Hypothesis: We hypothesize that SGLT2 colocalizes on both Glu and GABAergic nTS neurons but increases due to a high salt diet. Methods: Male Dahl-Salt Sensitive Rats were fed a normal 0.4% salt diet (NSD) for 2 weeks and randomly placed to a NSD or a high 4% salt diet (HSD) for 7 weeks. After 9 weeks total, the brainstem section containing the nTS of each rat was isolated at 9AM. Goals/Aims: We used immunohistochemistry to identify either Glu (CAMKII) and GABAergic (GAD67) and localize SGLT2. Using a confocal microscope, a Z-stack image was compiled at 40x magnification and colocalization analysis was completed to determine if SGLT2 is found greater on Glu or GABAergic neurons. Results: We examined 3 areas within the nTS: caudal, rostral, and mid (area postrema, a region where cardiorespiratory afferent information is received). We first found that SGLT2 colocalizes to different extents on both Glu and GABAergic nTS neurons. We used Pearson’s R correlation to determine the strength and linear relationship between cell type and SGLT2. In GABAergic neurons, we found a positive correlation (r=0.319) in NSD, which decreased significantly in HSD (p=0.013, t-test) in the nTS at area postrema. Conversely, within the caudal nTS, SGLT2 colocalizes to a greater extent on Glu neurons in HSD (r=0.434) and lower in NSD (r=0.308), (p=0.04, t-test). Conclusions: In our model, we find that SGLT2 colocalization with Glu and GABAergic neurons is present within the nTS in a location-dependent manner, which may affect its function in the development of hypertension. Further experiments exploring the SGLT2 within normotensive and hypertensive rats may better reveal the function of the protein within the nTS.

Abstract P141: Salt tips the balance of tryptophan metabolism between host and gut microbiota in a sex specific manner

Introduction: Tryptophan (Trp) diet attenuates hypertension in male Dahl salt sensitive (S) rats with unknown mechanisms. Kynurenine (a vasodilator) and serotonin (mixed effects on blood pressure) are host-derived Trp metabolites while indole is produced exclusively by the gut microbiota. We have previously shown that the gut microbiota of male, but not female, S rats is susceptible to salt, causing a higher indole level that elevated blood pressure. In this study, we reported the sex differences in all Trp metabolites with a focus on the impact of salt and the mechanisms by which female gut microbiota is more resilient. Methodology: S rats of both sexes (10–13-week-old, n = 4-7/group) were maintained on either a low-salt (0.3 % NaCl, LS) or high-salt (2 % NaCl, HS) diet for 3 weeks. Trp metabolites were analyzed in serum and cecum by LC-MS/MS. To investigate if estrogen shape gut microbiota, gonadectomy was performed on 7-week-old Spontaneously Hypertensive Rats (SHR) of both sexes (n=5/group). β-estradiol (2.8 mg/kg) was administered subcutaneously for 12 weeks before fecal 16S rRNA gene sequencing. Results: Females had elevated levels of kynurenine (Kyn) compared to male S rats on both LS (5.5 ± 1.4 vs. 2.1 ± 0.5 µM; P <0.05) and HS (7 ± 1.3 vs. 4.2 ± 1 µM; P <0.05). Kyn metabolites including kynurenic acid, 3-hydroxykynurenine, 3-hydroxyanthranilic acid were elevated in the serum of females compared with males, and HS amplified this difference (all P <0.05). Further, HS significantly decreased the serum serotonin levels in both sexes ( P <0.01 in females, P <0.0001 in males). Intriguingly, HS increased cecal indole levels in male ( P <0.05), but not in female rats. Microbiota analysis of gonadectomized (GDX) SHR rats revealed (1) Significant differences in β-diversity between intact vs. GDX rats ( P <0.01), and between GDX male vs. female rats ( P =0.01); (2) Estrogen supplementation eliminates the gut microbiota differences between GDX females and males ( P =0.18). Conclusion: We demonstrated sex differences in Trp metabolism in response to high salt. Further, estrogen masked the microbial differences between GDX male and female SHR rats. In summary, salt led to an elevated Trp-Kyn metabolism in female host, and an enhanced Trp-indole metabolism in male gut microbiota, which could be due to the estrogen’s effect on shaping gut microbiota. Thus, the Trp metabolism in salt-sensitive hypertension is regulated in a sex-specific manner.

Abstract P451: Effects of Perivascular Adipose Tissue on Arterial Stiffness in an Adiposity-Driven Hypertension Model: A Longitudinal Study

Perivascular adipose tissue (PVAT) is crucial for maintaining arterial function under healthy conditions. This research aims to test the hypothesis that the effect of PVAT on arterial stiffness is lessened as hypertension progresses in an animal model of adiposity-driven hypertension. We examined thoracic aorta samples from male Dahl Salt-Sensitive rats subjected to control (10% fat-CD) and high-fat (60% fat-HFD) diets for 16 and 24 weeks. Systolic blood pressure was significantly higher in HFD rats only at 24 weeks as compared to CD. Uniaxial mechanical tests were then conducted on aorta samples with and without PVAT. Arterial wall stiffness at 20% stretch (matching in vivo measures) was increased but not significantly higher in animals fed a HFD compared to those on a CD at 16 weeks but not 24 weeks. Surprisingly, the calculated stiffness of -PVAT samples significantly decreased by 24 weeks in both groups. Stiffness was reduced at both time points in +PVAT samples compared to -PVAT, but the beneficial effect of PVAT is diminished at 24 weeks. Because lower stretches may be important as hypertension progresses. We also measured stiffness at stretches <10%. Here, the addition of PVAT lost all beneficial effects on stiffness at 24 weeks. These results suggest that while PVAT has a beneficial effect on arterial stiffness in adiposity-driven hypertension, its effectiveness decreases with aging.

Puerarin Alleviates Blood Pressure via Inhibition of ROS/TLR4/NLRP3 Inflammasome Signaling Pathway in the Hypothalamic Paraventricular Nucleus of Salt-Induced Prehypertensive Rats.

Puerarin is an isoflavone compound isolated from the roots of a leguminous plant, the wild kudzu. Various functional activities of this compound in multiple diseases have been reported. However, the effect and mechanism of puerarin in improving blood pressure remain non-elucidated. The current study was designed to assess the preventive effects of puerarin on the onset and progression of hypertension and to verify the hypothesis that puerarin alleviates blood pressure by inhibiting the ROS/TLR4/NLRP3 inflammasome signaling pathway in the hypothalamic paraventricular nucleus (PVN) of salt-induced prehypertensive rats. Male Dahl salt-sensitive rats were fed low NaCl salt (3% in drinking water) for the control (NS) group or 8% (HS) to induce prehypertension. Each batch was divided into two group and treated by bilateral PVN microinjection with either artificial cerebrospinal fluid or puerarin through a micro-osmotic pump for 6 weeks. The mean arterial pressure (MAP) was recorded, and samples were collected and analyzed. We concluded that puerarin significantly prevented the elevation of blood pressure and effectively alleviated the increase in heart rate caused by high salt. Norepinephrine (NE) in the plasma of salt-induced prehypertensive rats also decreased upon puerarin chronic infusion. Additionally, analysis of the PVN sample revealed that puerarin pretreatment decreased the positive cells and gene level of TLR4 (Toll-like receptor 4), NLRP3, Caspase-1 p10, NOX2, MyD88, NOX4, and proinflammatory cytokines in the PVN. Puerarin pretreatment also decreased NF-κBp65 activity, inhibited oxidative stress, and alleviated inflammatory responses in the PVN. We conclude that puerarin alleviated blood pressure via inhibition of the ROS/TLR4/NLRP3 inflammasome signaling pathway in the PVN, suggesting the therapeutic potential of puerarin in the prevention of hypertension.

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.

Sex-Dependency of T Cell-Induced Salt-Sensitive Hypertension and Kidney Damage.

It is established that the immune system, namely T cells, plays a role in the development of hypertension and renal damage in male Dahl salt-sensitive (SS) rats, but far less is known about this relationship in females. Rats with genetically deleted T cells via CD247 gene mutation on the Dahl SS background (SSCD247-/-) were utilized to interrogate the effect of sex and T cells on salt sensitivity. We assessed the hypertensive and kidney injury phenotypes in male versus female SS and SSCD247-/- rats challenged with 3 weeks of high salt (4.0% NaCl). Differences in T cell activation genes were examined in renal T cells from male and female SS rats, and a sex-specific adoptive transfer was performed by injecting male or female splenocytes into either male or female SSCD247-/- recipients to determine the potential contribution of T cell sex. The lack of functional T cells in SSCD247-/- rats significantly reduced salt-induced hypertension and proteinuria in both sexes, although SSCD247-/- females exhibited greater protection from kidney damage. Adoptive transfer of either Dahl SS male or female splenocytes into SSCD247-/- male recipients exacerbated hypertension and proteinuria compared with controls, while in SSCD247-/- female recipients, exacerbation of disease occurred only upon transfer of male, but not female, SS splenocytes. The absence of T cells in the SSCD247-/- normalized sex differences in blood pressure, though sex differences in renal damage persisted. Splenocyte transfer experiments demonstrated that salt sensitivity is amplified if the sex of the T cell or the recipient is male.

THE SENOLYTIC QUERCETIN PREVENTS AGE-ASSOCIATED VASCULAR STIFFENING, REDUCES BLOOD PRESSURE AND IMPROVES COGNITIVE FUNCTION IN DAHL SALT-SENSITIVE RATS CONSUMING A NORMAL SALT DIET

Objective: Quercetin (QRC), a flavonoid with anti-oxidant, anti-inflammatory and senolytic effects, reduced BP in salt-induced hypertension in Dahl salt-sensitive (DSS) rats. The increases in central arterial stiffness (CAS) and blood pressure (BP) occur, as manifestation of early vascular aging (EVA), in DSS rats even consuming a normal salt (NS) diet, moreover, EVA is associated with cognitive impairment in humans. We hypothesized that as age advances, a decline in cognitive function will accompany EVA in DSS rats consuming a NS diet, and that QRC will not only retard EVA, but also will improve cognitive function. Design and method: A NS diet (0.5% NaCl), supplemented with QRC (100 mg/kg BW/day) was administered to male DSS rats at 6-mo of age (n=24) for a 3-month period. DSS without QRC supplementation served as a control group (n=12). Systolic and diastolic BP (SBP, DBP), pulse wave velocity (PWV, an index of CAS), echocardiography, and a visuospatial attention test were assessed prior to and following QRC treatment. Results: At 6-mo of age none of the measured parameters differ between control and QRC rats. During aging from 6- to 9-mo in the control group, SBP, DBP and PWV significantly increased, ESLV.vol showed a statistically borderline trend to increase, HR and EF showed a statistically borderline trend to decrease, and attention behavior did not change. QRC treatment reduced SBP, PWV, and ESLV.vol, and increased EF and attention (Table). The QRC effect to increase the number of correct attention test responses was inversely correlated with PWV (Pearson r=-0.420, P=0.02) but not with BP or cardiac parameters, indicating that CAS but not BP is linked to cognitive function. Conclusions: QRC reduces EVA and improves cognition assessed as an attention behavior. Although both BP and CAS are manifestations of EVA, a positive association between attention decline with PWV, but not BP, suggests that CAS is linked to cognitive function via mechanisms that are not directly related to BP. Further studies are required to determine whether the effects of QRC on EVA in DSS rats, consuming a NS diet, are linked to its anti-oxidant, anti-inflammatory or senolytic properties.

A cell atlas of thoracic aortic perivascular adipose tissue: a focus on mechanotransducers.

Perivascular adipose tissue (PVAT) is increasingly recognized for its function in mechanotransduction. However, major gaps remain in our understanding of the cells present in PVAT, as well as how different cells contribute to mechanotransduction. We hypothesized that snRNA-seq would reveal the expression of mechanotransducers, and test one (PIEZO1) to illustrate the expression and functional agreement between single-nuclei RNA sequencing (snRNA-seq) and physiological measurements. To contrast two brown tissues, subscapular brown adipose tissue (BAT) was also examined. We used snRNA-seq of the thoracic aorta PVAT (taPVAT) and BAT from male Dahl salt-sensitive (Dahl SS) rats to investigate cell-specific expression mechanotransducers. Localization and function of the mechanostransducer PIEZO1 were further examined using immunohistochemistry (IHC) and RNAscope, as well as pharmacological antagonism. Approximately 30,000 nuclei from taPVAT and BAT each were characterized by snRNA-seq, identifying eight major cell types expected and one unexpected (nuclei with oligodendrocyte marker genes). Cell-specific differential gene expression analysis between taPVAT and BAT identified up to 511 genes (adipocytes) with many (≥20%) being unique to individual cell types. Piezo1 was the most highly, widely expressed mechanotransducer. The presence of PIEZO1 in the PVAT but not the adventitia was confirmed by RNAscope and IHC in male and female rats. Importantly, antagonism of PIEZO1 by GsMTX4 impaired the PVAT's ability to hold tension. Collectively, the cell compositions of taPVAT and BAT are highly similar, and PIEZO1 is likely a mechanotransducer in taPVAT.NEW & NOTEWORTHY This study describes the atlas of cells in the thoracic aorta perivascular adipose tissue (taPVAT) of the Dahl-SS rat, an important hypertension model. We show that mechanotransducers are widely expressed in these cells. Moreover, PIEZO1 expression is shown to be restricted to the taPVAT and is functionally implicated in stress relaxation. These data will serve as the foundation for future studies investigating the role of taPVAT in this model of hypertensive disease.

Canagliflozin attenuates kidney injury, gut-derived toxins, and gut microbiota imbalance in high-salt diet-fed Dahl salt-sensitive rats

Purpose To investigate the effects of canagliflozin (20 mg/kg) on Dahl salt-sensitive (DSS) rat gut microbiota and salt-sensitive hypertension-induced kidney injury and further explore its possible mechanism. Methods Rats were fed a high-salt diet to induce hypertension and kidney injury, and physical and physiological indicators were measured afterwards. This study employed 16S rRNA sequencing technology and liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based metabolic profiling combined with advanced differential and association analyses to investigate the correlation between the microbiome and the metabolome in male DSS rats. Results A high-salt diet disrupted the balance of the intestinal flora and increased toxic metabolites (methyhistidines, creatinine, homocitrulline, and indoxyl sulfate), resulting in severe kidney damage. Canagliflozin contributed to reconstructing the intestinal flora of DSS rats by significantly increasing the abundance of Corynebacterium spp., Bifidobacterium spp., Facklamia spp., Lactobacillus spp., Ruminococcus spp., Blautia spp., Coprococcus spp., and Allobaculum spp. Moreover, the reconstruction of the intestinal microbiota led to significant changes in host amino acid metabolite concentrations. The concentration of uremic toxins, such as methyhistidines, creatinine, and homocitrulline, in the serum of rats was decreased by canagliflozin, which resulted in oxidative stress and renal injury alleviation. Conclusion Canagliflozin may change the production of metabolites and reduce the level of uremic toxins in the blood circulation by reconstructing the intestinal flora of DSS rats fed a high-salt diet, ultimately alleviating oxidative stress and renal injury.

Hypoxia-inducible factor-prolyl hydroxylase inhibitor Roxadustat (FG-4592) reduces renal fibrosis in Dahl salt-sensitive rats.

Although hypoxia-inducible factor-prolyl hydroxylase (HIF-PH) inhibitors have been developed for the treatment of renal anemia, their effects on cardiac and renal dysfunction remain unknown. We previously reported on Dahl salt-sensitive rats, in a rat model of salt-sensitive hypertension, that exhibited anemia and impaired expression of duodenal iron transporters after the development of hypertensive cardiac and renal dysfunction. Therefore, we investigated the effects of Roxadustat (FG-4592), an HIF-PH inhibitor, on anemia, iron regulation, and cardiac and renal dysfunction in Dahl salt-sensitive rats. Six-week-old male Dahl salt-sensitive rats were fed a normal or high-salt diet for 8 weeks. A further subset of Dahl salt-sensitive rats, that were fed a high-salt diet, was administered Roxadustat for 8 weeks. Dahl salt-sensitive rats fed a high-salt diet developed hypertension, cardiac and renal dysfunction, and anemia after 8 weeks of feeding. Roxadustat increased hemoglobin and serum erythropoietin levels in Dahl salt-sensitive rats fed a high-salt diet. With regard to the iron-regulating system, Roxadustat lowered hepatic hepcidin gene expression and increased the gene expression of duodenal iron transporters, such as cytochrome b and divalent metal transporter 1 , in Dahl salt-sensitive rats fed a high-salt diet. Roxadustat did not affect the development of hypertension and cardiac hypertrophy in Dahl salt-sensitive rats with a high-salt diet; however, Roxadustat treatment attenuated renal fibrosis in these rats. Roxadustat ameliorated anemia with affecting the gene expression of the iron-regulating system, and did not affect cardiac hypertrophy but attenuated renal fibrosis in Dahl salt-sensitive rats fed a high-salt diet.

Transcriptomic changes in glomeruli in response to a high salt challenge in the Dahl SS rat.

Salt sensitivity impacts a significant portion of the population and is an important contributor to the development of chronic kidney disease. One of the significant early predictors of salt-induced damage is albuminuria, which reflects the deterioration of the renal filtration barrier: the glomerulus. Despite significant research efforts, there is still a gap in knowledge regarding the molecular mechanisms and signaling networks contributing to instigating and/or perpetuating salt-induced glomerular injury. To address this gap, we used 8-wk-old male Dahl salt-sensitive rats fed a normal-salt diet (0.4% NaCl) or challenged with a high-salt diet (4% NaCl) for 3 wk. At the end of the protocol, a pure fraction of renal glomeruli obtained by differential sieving was used for next-generation RNA sequencing and comprehensive semi-automatic transcriptomic data analyses, which revealed 149 differentially expressed genes (107 and 42 genes were downregulated and upregulated, respectively). Furthermore, a combination of predictive gene correlation networks and computational bioinformatic analyses revealed pathways impacted by a high salt dietary challenge, including renal metabolism, mitochondrial function, apoptotic signaling and fibrosis, cell cycle, inflammatory and immune responses, circadian clock, cytoskeletal organization, G protein-coupled receptor signaling, and calcium transport. In conclusion, we report here novel transcriptomic interactions and corresponding predicted pathways affecting glomeruli under salt-induced stress.NEW & NOTEWORTHY Our study demonstrated novel pathways affecting glomeruli under stress induced by dietary salt. Predictive gene correlation networks and bioinformatic semi-automatic analysis revealed changes in the pathways relevant to mitochondrial function, inflammatory, apoptotic/fibrotic processes, and cell calcium transport.

Abstract 14993: Empagliflozin Exerts a Sympathoinhibitory Effect and Ameliorates Cardiorenal Injuries in a Rat Model of Hypertensive Heart Failure

Background: SGLT2 inhibitors may have protective effects on cardiac and renal injuries; however, their mechanisms remain unclear. Sympathetic activation has a crucial role in cardio/renal injuries. This study aimed to investigate whether empagliflozin can inhibit cardio/renal injuries through sympathoinhibition in a rat model of hypertensive heart failure. Methods: Male Dahl salt-sensitive rats were divided into low-salt (LS) and high-salt (HS) diet groups from 6 weeks of age. The rats in HS group were assigned to vehicle (HS-VEH) or empagliflozin (HS-EMPA) treatment group at 8 weeks. The effect of empagliflozin on central sympathetic regulation and cardio/renal injuries was assessed at 12 weeks (pre-heart failure phase), while its protective effect on heart failure was evaluated at 15 weeks in another cohort. Results: At 12 weeks , systolic blood pressure was similarly elevated in HS-VEH and HS-EMPA. Plasma norepinephrine levels were increased in HS-VEH compared to LS, and were suppressed in HS-EMPA, which was consistent with the neuronal activity of pre-sympathetic neurons in the brain. Increased left ventricular (LV) weight/body weight, plasma creatinine levels, renal fibrosis, and glomerular sclerosis in HS-VEH were attenuated in HS-EMPA. At 15 weeks , the high salt-induced blood pressure elevation was similar between HS-VEH and HS-EMPA. Although LV hypertrophy did not significantly differ, both decreased echocardiographic LV fractional shortening and increased lung weight observed in HS-VEH were attenuated in HS-EMPA. Elevated plasma creatinine and norepinephrine levels in HS-VEH were also attenuated in HS-EMPA. Conclusion: Empagliflozin inhibits sympathoexcitation and ameliorates cardio/renal injuries without lowering blood pressure in the hypertensive heart failure model. This sympathoinhibitory effect of empagliflozin observed in the pre-heart failure phase might contribute to the attenuation of heart failure progression.

Abstract 14951: Input From Renal Afferent Nerves Contributes to Hypertensive Heart Failure With Preserved Systolic Function Through Baroreflex Impairment and Sympathoexcitation

Introduction: Activation of renal afferent nerves, which can be caused by hypertensive renal damage, increases sympathetic output. The nucleus tractus solitarius (NTS) receives the inputs from baroreflex afferent nerves as well as renal afferent nerves. Baroreflex dysfunction leads to increased left ventricular (LV) end-diastolic pressure even in the heart with preserved systolic function. The GABAergic inhibitory system in the NTS blunts the signals from baroreflex afferent nerves, resulting in increased sympathetic output. Hypothesis: Input from renal afferent nerves contributes to hypertensive heart failure with preserved systolic function through baroreflex impairment and sympathoexcitation mediated by increased GABAergic inhibition in the NTS. Methods and Results: Male Dahl salt-sensitive rats with 8% high-salt diet from 7 weeks of age develop hypertensive HFpEF at 19 weeks. These rats underwent sham surgery (Sham), selective afferent renal denervation (ARDN), or additionally dissection of baroreflex afferent nerves (ARDN+BAROX) at 9 weeks. As controls, rats underwent sham surgery with a low-salt diet. The rats were evaluated at 19 weeks. Systolic blood pressure was similarly elevated in the three groups on high-salt diet. Impaired LV diastolic function assessed by echocardiography and pressure-volume loop analysis in Sham was recovered in ARDN group, while systolic function was preserved in all the group. Decreased baroreflex sensitivity and increased sympathetic output in Sham were attenuated in ARDN group. Activity of GABAergic neurons in the NTS evaluated by immunostaining was increased in Sham compared to controls, and was attenuated in ARDN group. All of the changes in ARDN were cancelled in ARDN+BAROX group. Conclusion: Input from renal afferent nerves contributes to hypertensive heart failure with preserved systolic function through baroreflex impairment and sympathoexcitation along with increased GABAergic inhibition in the NTS.

Pathological mechanism of heart failure with preserved ejection fraction in rats based on iTRAQ technology.

Heart failure with preserved ejection fraction (HFpEF) is a public health problem worldwide. Treatments for the patients with HFpEF are not satisfactory because there is no unified understanding of the pathological mechanism of HFpEF. This study aims at investigating the potential pathological mechanism for the effective diagnosis and treatment of HFpEF. Ten adult male Dahl salt sensitive rats (180-200 g) were divided into control and model groups. The rats in model group were fed with high salt diet (8% NaCl) to induce HFpEF for this comparative study. Behavioral changes, biochemical parameters, and histopathological changes of the rats were detected. iTRAQ technology combined with bioinformatics analysis was employed to study the differentially expressed proteins (DEPs) and their enrichment in signaling pathways. Echocardiography detection showed decreased LVEF, indicating impaired cardiac function (P<0.01), increased LVPWd, indicating ventricular wall hypertrophy (P<0.05), prolonged duration of IVRT and decreased E/A ratio, indicating diastolic dysfunction (P<0.05) of the rats in model group. 563 DEPs were identified in the rats of both groups, with 243 up-regulated and 320 down-regulated. The expression of PPAR signaling pathway in the rats of model group was down-regulated, with PPARα most significantly decreased (91.2%) (P<0.01), PPARγ obviously decreased (63.60%) (P<0.05), and PPARβ/δ decreased (45.33%) (P<0.05). The DEPs enriched in PPAR signaling pathway were mainly related to such biological processes as fatty acid beta-oxidation, such cellular components as peroxisome, and such molecular functions as lipid binding. NaCl high salt diet is one of the factors to increase the incidence of HFpEF in rats. PPARα, PPARγ and PPAR β/δ might be the targets of HFpEF. The findings may provide a theoretical basis for the treatment of HFpEF in clinical practice.

Input from afferent renal nerves is involved in progression of hypertensive heart failure through central vasopressin-mediated sympathoexcitation

Introduction: Paraventricular nucleus of hypothalamus (PVN), a control center of sympathetic outflow, receives the input from afferent renal nerves. Although acute stimulation of afferent renal nerves increases central sympathetic outflow, it remains unclear if the renal afferents contribute to progression of heart failure. Acute stimulation of afferent renal nerves was reported to increase plasma vasopressin concentration. In addition, central dendritic release of vasopressin by activation of vasopressin-expressing neurons located in the PVN may cause sympathoexcitation via stimulation of neighboring presympathetic neurons in the PVN. Therefore, we hypothesized that the chronic input from afferent renal nerves contributes to sympathoexcitation and hypertensive cardiac dysfunction along with activation of vasopressin-expressing neurons and presympathetic neurons in the PVN. Methods: Male Dahl salt-sensitive rats were fed low salt diet (LS) or 8% high salt diet from 6 weeks of age, and the high salt-fed rats were allocated to selective afferent renal denervation (HS-ARDN) or sham surgery (HS-Sham) at 9 weeks. The effect of ARDN on central sympathetic regulation was investigated at 12 weeks (pre-heart failure phase). In another cohort, its effect on cardiac phenotypes was evaluated at 16 weeks (heart failure phase). Results: At 12 weeks, plasma norepinephrine level was increased in HS-Sham compared to LS and was attenuated by ARDN (LS 188.7±16.5 pg/ml, HS-Sham 563.8±82.7 pg/ml, HS-ARDN 327.4±50.2 pg/ml; n=15, 12, 14; p&lt;0.05). The c-Fos expression, a marker of neuronal activity, in presympathetic neurons in the PVN was also increased in HS-Sham and attenuated in HS-ARDN, which was in parallel with the c-Fos expression in vasopressin-expressing neurons in the PVN. At 16 weeks, the decrease of left ventricular (LV) fractional shortening (HS-Sham 25.9±1.7 % vs LS 46.7±0.6 %, n=9, 9; p&lt;0.01) was improved in HS-ARDN (42.2±1.2 %, n=7; p&lt;0.01 vs HS-Sham). LV weight and lung weight were significantly increased in HS-Sham compared to LS (LV weight/body weight: 4.39±0.19 mg/g vs 2.53±0.03 mg/g; lung weight/body weight: 5.29±0.37 mg/g vs 3.55±0.05 mg/g; p&lt;0.05), and these changes were attenuated in HS-ARDN (LV weight/body weight: 3.73±0.14 mg/g; lung weight/body weight: 4.17±0.11 mg/g; p&lt;0.05 vs HS-Sham). Histological LV fibrosis and fibrosis marker expression, such as connective tissue growth factor, in LV were significantly attenuated in HS-ARDN compared to HS-Sham. ARDN did not significantly impact the high salt-induced blood pressure elevation in both cohorts. Conclusion: The input from afferent renal nerves contributes to cardiac dysfunction, probably via the central vasopressin-mediated sympathoexcitation even without blood pressure changes in hypertensive heart failure model. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Salt-dependent Role of Human Gut Commensal Coprococcus comes in Blood Pressure and Inflammation

Introduction: We previously identified the effect of Coprococcus come, a common human gut microflora, on blood pressure regulation due to its metabolism of angiotensin-converting enzyme inhibitors (ACEi). Despite being reported as a butyrate-producer, there are no studies on C. comes in its direct role in hypertension and inflammation. Importantly, C. comes was enriched in the African American hypertensive population, almost three-fourths of whom were found salt sensitive. Therefore, we hypothesized that C. comes increased blood pressure and associated systemic inflammation in a salt-dependent manner. Methods: Once per day for 13 consecutive days, male Dahl salt-sensitive (S) rats (N=7-8/group) were administered 109 CFU of C. comes via oral gavage on both low and high salt diet. Systolic blood pressure, diastolic blood pressure, and mean arterial pressure were recorded on day 13 by radio-telemetry. On day 14, spleen (the key organ in systemic inflammation) and kidney (the key organ in salt hypertension) were collected for analyses of inflammatory markers by real time PCR and flow cytometry. Results: In low salt rats, the administration of C. comes did not significantly change blood pressures, but resulted in immunosuppression in the spleen, as evidenced by decreased inflammatory markers Il6 (0.79 fold, p&lt;0.05) and Il17rc (0.74 fold, p&lt;0.001). In contrast, there were significant increases in the S rats on high salt+ C. comes vs high salt in systolic blood pressure (183.2 vs 179.9 mmHg, p&lt;0.01), diastolic blood pressure (130.5 vs 127.1 mmHg, p&lt;0.01), and mean arterial pressures (156.7 vs 151.8 mmHg, p&lt;0.0001) during nighttime, but not daytime. The administration of C. comes in high salt rats led to increased inflammation in the spleen, as measured by the percentage of CD11b/c+ cells (high salt+ C. comes 2.9% vs high salt 2.1% p&lt;0.05) and CD68+ cells (high salt+ C. comes 0.28% vs high salt 0.22% p&lt;0.05), as well as in the kidney, as measured by inflammatory markers Il1b (4.4 fold, p&lt;0.05), Il17rc (8.8 fold, p&lt;0.05), and Tnf (1.67 fold, p&lt;0.05). Conclusion: C. comes, an ACEi degrader, suppressed splenic inflammation in the male S rats on a low salt diet without impacting blood pressure. However, on high salt diet, C. comes administration resulted in increased splenic and renal inflammation as well as increased blood pressure in the male S rats. These findings support the pathogenic role of C. comes in salt-induced hypertension, providing evidence for targeting C. comes in blood pressure control. This study is funded by NIH R21AG079357 and AHA CDA 852969. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diet modification reverses diastolic dysfunction in rats with heart failure and preserved ejection fraction.

Dahl Salt-Sensitive (DSS) rats develop heart failure with preserved ejection fraction (HFpEF) when fed a high-salt (8 % NaCl) diet. Hypertension-induced inflammation and subsequent ventricular fibrosis are believed to underlie the development of HFpEF. We investigated the role of diet modification in the progression of HFpEF using male DSS rats, fed either a high-salt diet from7 weeks of age to induce HFpEF, ora normal-salt (0.3% NaCl) diet as controls. After echocardiographic confirmation of diastolic dysfunction at 14-15 weeks of age along with HF manifestations, the HFpEF rats were randomly assigned to either continue a high-salt diet or switch to a normal-salt diet for an additional 4 weeks. HFpEF rats with diet modification showed improved diastolic function (reduced E/E' ratio in echocardiogram), increased functional capacity (increased treadmill exercise distance), and reduced pulmonary congestions (lung/body weight ratio), compared to high-salt-fed HFpEF rats. Systolic blood pressure remained high (~200 mmHg), and ventricular hypertrophy remained unchanged. Ventricular arrhythmia inducibility (100 % inducible) and corrected QT interval (on ECG) did not change in HFpEF rats after diet modification. HFpEF rats with diet modification showed prolonged survival and reduced ventricular fibrosis (Masson's trichrome staining) compared to high-salt-fed HFpEF rats. Hence, the modification of diet (from high-salt to normal-salt diet) reversed HFpEF phenotypes without affecting blood pressure or ventricular hypertrophy.

Altered Neuronal Discharge in the Organum Vasculosum of the Lamina Terminalis Contributes to Dahl Salt-Sensitive Hypertension.

Salt-sensitive hypertension in humans and experimental models is associated with higher plasma and cerebrospinal fluid sodium chloride (NaCl) concentrations. Changes in extracellular NaCl concentrations are sensed by specialized neurons in the organum vasculosum of the lamina terminalis (OVLT). Stimulation of OVLT neurons increases sympathetic nerve activity (SNA) and arterial blood pressure (ABP), whereas chronic activation produces hypertension. Therefore, the present study tested whether OVLT neuronal activity was elevated and contributed to SNA and ABP in salt-sensitive hypertension. Male Dahl salt-sensitive (Dahl S) and Dahl salt-resistant (Dahl R) rats were fed 0.1% or 4.0% NaCl diets for 3 to 4 weeks and used for single-unit recordings of OVLT neurons or simultaneous recording of multiple sympathetic nerves during pharmacological inhibition of the OVLT. Plasma and cerebrospinal fluid Na+ and Cl- concentrations were higher in Dahl S rats fed 4% versus 0.1% or Dahl R rats fed either diet. In vivo single-unit recordings revealed a significantly higher discharge of NaCl-responsive OVLT neurons in Dahl S rats fed 4% versus 0.1% or Dahl R rats. Interestingly, intracarotid infusion of hypertonic NaCl evoked greater increases in OVLT neuronal discharge of Dahl S versus Dahl R rats regardless of NaCl diet. The activity of non-NaCl-responsive OVLT neurons was not different across strain or diets. Finally, inhibition of OVLT neurons by local injection of the gamma-aminobutyric acid agonist muscimol produced a greater decrease in renal SNA, splanchnic SNA, and ABP of Dahl S rats fed 4% versus 0.1% or Dahl R rats. A high salt diet activates NaCl-responsive OVLT neurons to increase SNA and ABP in salt-sensitive hypertension.

PS-BPB04-4: RENAL DENERVATION IMPROVES THE SURVIVAL RATE VIA A REDUCTION IN HEART RATE IN HIGH SALT-FED DAHL SALT-SENSITIVE RATS

Objective: We reported that high salt intake decreases heart rates via a reduction in renal sympathetic activity in normal Sprague Dawley rats to compensate for high salt-driven catabolism. We also revealed that Dahl salt-sensitive (DSS) rats cannot reduce heart rates during high salt intake because of the abnormal activation of renal sympathetic nerve activity. It is known that high heart rates are negatively associated with life span. Therefore, we hypothesized that renal denervation ontributes to the long life span in high salt-fed DSS rats. Design and Method: (Experiment 1) We performed renal denervation (n = 9) or sham operation (n = 9) on male DSS rats at 4 weeks of age. At the same time, we implanted a radiotelemetry transmitter for blood pressure measurements in the above rats (sham (n = 4), renal denervation (n = 5)). After two weeks of recovery, we started feeding them a 4% NaCl diet with 0.9% NaCl water to drink (HS). We observed the survival rates, blood pressure, and heart rates. (Experiment 2) Male, 4-week-old DSS rats were divided into three groups: i) sham operation + 0.2% NaCl diet with tap water to drink, ii) sham operation + HS, and iii) renal denervation + HS (n = 6 per group). At 2 weeks post-surgery, the rats were fed the respective diets for 6 weeks. We weekly measured systolic blood pressure by a tail-cuff method. After 6 weeks of observation, we harvested their skin and skinned carcass to measure their body. Results: The renal-denervated DSS rats showed a higher survival rate compared to the sham-operated DSS rats (p &lt; 0.05). While renal denervation showed a decrease in heart rate, there was no significant decrease in mean arterial pressure throughout the experimental period. High salt intake significantly increased body Na content (mmol/g dry weight), whereas renal denervation did not alter body Na content in DSS rats. Conclusion: Renal denervation improved the survival rate in high salt-fed Dahl salt-sensitive rats, which may be mediated by a reduction in heart rate, but not body Na content nor blood pressure.