PVAT-conditioned media from Dahl S rats on high fat diet promotes inflammatory cytokine secretion by activated T cells prior to the development of hypertension.

There is considerable evidence that the immune system plays a role in hypertension, however this role is not fully characterized. Our previous studies demonstrated that mesenteric perivascular adipose tissue (mPVAT) harbors a large T cell population, which is a cell type identified as contributing to hypertension. In the present study, we tested the hypothesis that soluble mediators in mPVAT influence T cell function just prior to the development of hypertension. Toward this end, we utilized a unique model of hypertension in which Dahl S rats on a high fat (HF) diet develop hypertension. We found that conditioned media (CM) from mPVAT from healthy Dahl S rats on control diet buffers T cell activation, however, mPVAT-CM from Dahl S rats on a HF diet markedly increased inflammatory cytokine induction (IFNγ, GM-CSF and IL-17a) by activated T cells. These cytokines are known to promote activation of macrophages and neutrophils, among other effects. Conversely, the anti-inflammatory cytokine, IL-10, was not different between the groups, suggesting the effect is selective for inflammatory cytokines. Furthermore, we conducted bulk RNA-seq on activated T cells cultured in mPVAT-CM from Dahl S rats on either control (CTL) or HF diet for 10 weeks. In accordance with the cytokine analysis, mPVAT-CM from HF diet-fed rats significantly upregulated many genes associated with IFNγ/IL-17 induction, whereas Th2/Treg-associated genes were downregulated. Taken together, these data strongly suggest soluble mediators from mPVAT influence T cell inflammatory status and may promote Th1/Th17 differentiation preceding the development of hypertension triggered by HF diet.

Genetically conditioned interaction among microRNA-155, alpha-klotho, and intra-renal RAS in male rats: Link to CKD progression.

Incident chronic kidney disease (CKD) varies in populations with hypertension of similar severity. Proteinuria promotes CKD progression in part due to activation of plasminogen to plasmin in the podocytes, resulting in oxidative stress-mediated injury. Additional mechanisms include deficiency of renal alpha-klotho, that inhibits Wnt/beta-catenin, an up regulator of intra-renal renin angiotensin system (RAS) genes. Alpha-klotho deficiency therefore results in upregulation of the intra-renal RAS via Wnt/beta-catenin. In hypertensive, Dahl salt sensitive (DS) and spontaneously hypertensive rats (SHR), we investigated renal and vascular injury, miR-155, AT1R, alpha-klotho, and TNF-α. Hypertensive high salt DS (DS-HS), but not SHR developed proteinuria, plasminuria, and glomerulosclerosis. Compared to DS low salt (DS-LS), in hypertensive DS-HS alpha-klotho decreased 5-fold in serum and 2.6-fold in kidney, whereas serum mir-155 decreased 3.3-fold and AT1R increased 52% in kidney and 77% in aorta. AT1R, alpha-klotho, and miR-155 remained unchanged in prehypertensive and hypertensive SHR. TNF-α increased by 3-fold in serum and urine of DS-HS rats. These studies unveiled in salt sensitive DS-HS, but not in SHR, a genetically conditioned dysfunction of the intermolecular network integrated by alpha-klotho, RAS, miR-155, and TNF-α that is at the helm of their end-organ susceptibility while plasminuria may participate as a second hit.

[Canagliflozin can improve cardiac function in HFpEF rats partly by regulating ferroptosis].

Objective: To explore the effects of canagliflozin on cardiac function and its regulation of ferroptosis in rats with heart failure with preserved ejection fraction (HFpEF). Methods: Thirty-two 7-week-old Dahl salt-sensitive rats were selected and randomly divided into four groups: the control group (fed with low-salt diet), the HFpEF group (fed with high-salt diet), the canagliflozin 20 group (fed with high-salt diet and 20 mg·kg-1·d-1 canagliflozin), and the canagliflozin 30 group (fed with high-salt diet and 30 mg·kg-1·day-1 canagliflozin). Body weight and blood pressure of the rats in each group were monitored. Metabolic cage tests were conducted at the10th week of the experiment, and echocardiography was performed at the 12th week, after which the rats were killed. Blood and left ventricular samples were collected. HE staining, Masson staining, Prussian blue iron staining, and reactive oxygen species staining were performed to observe the cardiomyocyte size and shape, degree of interstitial fibrosis, iron staining, reactive oxygen species production under optical microscope. The ultrastructure of cardiomyocytes was observed under electron microscope. Western blotting and real-time fluorescent quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to detect the expression levels of proteins and mRNA related to ferroptosis in left ventricular myocardial tissue of rats in each group. Results: After 1 week of adaptive feeding, all rats survived. Metabolic cage results showed that compared with control group, rats in the HFpEF group, canagliflozin 20 group and canagliflozin 30 group had more food intake, water intake and urine output, and lower body weight (all P<0.05). These changes were more pronounced in canagliflozin 20 group and canagliflozin 30 group than in HFPEF group, and only the body weight at the 12th week showed a statistically significant difference between canagliflozin 20 group and canagliflozin 30 group (P<0.05). The blood pressure of 6th week and 12th week, heart weight and left ventricular corrected mass of 12th week of rats in HFpEF group were higher than those in control group, canagliflozin 20 group and canagliflozin 30 group, while the ratio of early mitral valve peak velocity to late mitral valve peak velocity of 12th week was lower (all P<0.05). HE and Masson staining showed that compared to control group, the myocardial fibers in the left ventricular myocardial tissue of rats in HFpEF group were disordered, with larger cell diameter ((0.032±0.004) mm vs. (0.023±0.003) mm, P<0.05), irregular shape, obvious proliferation of interstitial collagen fibers, and higher collagen volume fraction (0.168±0.028 vs. 0.118±0.013, P<0.05). Compared with HFpEF group, rats in the canagliflozin 20 group and canagliflozin 30 had more orderly arranged myocardial fibers, more regular cardiomyocyte shape, smaller cell diameter, and lower collagen volume fraction (P<0.05). It was observed under electron microscopy that, compared to control group, most of the striated muscles in myocardial tissue of HFpEF group were broken, and the Z line and M line could not be clearly distinguished, some changes such as mitochondrial swelling, membrane thickening, cristae reduction or even disappearance occurred. In the canagliflozin 20 group and canagliflozin 30 group, the arrangement of striated muscles in the myocardial tissue of rats tended to be more regular, and the morphological changes of mitochondria were milder. Prussian blue iron staining results showed that the iron content in myocardial tissue of rats in HFpEF group was higher than that in control group, canagliflozin 20 group and canagliflozin 30 group. Reactive oxygen species staining results showed that the reactive oxygen species content in the myocardial tissue of rats in HFpEF group was higher than that of control group, canagliflozin 20 group and canagliflozin 30 group. Biochemical analysis of myocardial tissue showed that Fe2+ and malondialdehyde content in myocardial tissue of rats in HFpEF group were higher than those in control group, canagliflozin 20 group and canagliflozin 30 group, while glutathione content was lower (all P<0.05). Western blot and RT-qPCR detection results showed that compared to control group, rats in HFpEF group had higher expression levels of transferrin receptor 1 (protein relative expression level: 1.37±0.16 vs. 0.31±0.12), acyl-CoA synthetase long-chain family member 4 (protein relative expression level: 1.31±0.15 vs. 0.63±0.09) protein and mRNA, and lower expression levels of ferritin heavy chain 1 (protein relative expression level: 0.45±0.08 vs. 1.41±0.15) protein and mRNA (all P<0.05). There was no statistically significant difference in these indicators between canagliflozin 20 group and the canagliflozin 30 group (all P>0.05). There was no significant difference in levels of glutathione peroxidase 4 protein and mRNA expression in myocardial tissue of rats in four groups(P>0.05). Conclusion: Canagliflozin improves cardiac function in HFpEF rats by regulating the ferroptosis mechanism.

Irisin mitigates salt-sensitive hypertension via regulating renal AMPK-Rac1 pathway.

BACKGROUND: Irisin, as a myokine, plays a protective role against cardiovascular disease, including myocardial infarction, atherosclerosis and hypertension. However, whether irisin attenuates salt-sensitive hypertension and the related underlying mechanisms is unknown. METHODS: Male Dahl salt-resistant (DSR) and Dahl salt-sensitive (DSS) (12 weeks) rats were fed a high salt diet (8% NaCl) with or without irisin treatment by intraperitoneal injection for 8 weeks. RESULTS: Compared with DSR rats, DSS rats showed higher systolic blood pressure (SBP), impaired natriuresis and diuresis and renal dysfunction. In addition, it was accompanied by downregulation of renal p-AMPKα and upregulation of renal RAC1 and nuclear mineralocorticoid receptor (MR). Irisin intervention could significantly up-regulated renal p-AMPKα level and down-regulated renal RAC1-MR signal, thereby improving renal sodium excretion and renal function, and ultimately reducing blood pressure in DSS rats. Ex vivo treatment with irisin reduced the expression of RAC1 and nuclear MR in primary renal distal convoluted tubule cells from DSS rats and the effects of irisin were abolished by cotreatment of compound C (AMPK inhibitor), indicating that the regulation of RAC1-MR signals by irisin depended on the activation of AMPK. CONCLUSIONS: Irisin administration lowered salt-sensitive hypertension through regulating RAC1-MR signaling via activation of AMPK, which may be a promising therapeutic approach for salt-sensitive hypertension.

Renalase alleviates salt-induced kidney necroptosis and inflammation.

Recent evidence suggests that necroptosis may contribute to the development of kidney injury. Renalase is a novel secretory protein that exerts potent prosurvival and anti-inflammatory effects. We hypothesized that renalase could protect the kidney from salt-induced injury by modulating necroptosis. High salt and renalase treatments were administered to Dahl salt-sensitive (SS) rats, renalase knockout (KO) mice, and HK-2 cells. Furthermore, a cohort of 514 eligible participants was utilized to investigate the association between single nucleotide polymorphisms (SNPs) in the genes RIPK1, RIPK3, and MLKL, and the risk of subclinical renal damage (SRD) over 14 years. A high-salt diet significantly increased the expression of key components of necroptosis, namely RIPK1, RIPK3, and MLKL, as well as the release of inflammatory factors in SS rats. Treatment with recombinant renalase reduced both necroptosis and inflammation. In renalase KO mice, salt-induced kidney injury was more severe than in wild-type mice, but supplementation with renalase attenuated the kidney injury. In vitro experiments with HK-2 cells revealed high salt increased necroptosis and inflammation. Renalase exhibited a dose-dependent decrease in salt-induced necroptosis, and this cytoprotective effect was negated by the knockdown of PMCA4b, which is the receptor of renalase. Furthermore, the cohort study showed that SNP rs3736724 in RIPK1 and rs11640974 in MLKL were significantly associated with the risk of SRD over 14 years. Our analysis shows that necroptosis plays a significant role in the development of salt-induced kidney injury and that renalase confers its cytoprotective effects by inhibiting necroptosis and inflammation.

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

ABSTRACT: The hypothalamic paraventricular nucleus (PVN) plays a central role in regulating cardiovascular activity and blood pressure. We administered hydroxylamine hydrochloride (HA), a cystathionine-ß-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.

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

BACKGROUND: 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. PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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 effects of the atrial natriuretic peptide deficiency on renal cortical mitochondrial bioenergetics in the Dahl SS rat.

Atrial Natriuretic Peptide (ANP) plays an important role in blood pressure regulation. Low levels of ANP correlate with the development of salt-sensitive hypertension (SS-HTN). Our previous studies indicated that ANP deficiency exacerbated renal function decline in SS-HTN. In the heart and fat tissue, ANP was reported to affect lipid peroxidation and mitochondrial bioenergetics but the effects of ANP on mitochondrial function in the kidney are unexplored. We hypothesized that ANP deficiency in SS-HTN causes renal bioenergetic shift, leading to disruption of mitochondrial network and oxidative stress. To address the hypothesis, we placed Dahl SS wild-type (SSWT) and ANP knockout (SSNPPA-/-) rats on 4% NaCl high salt (HS) diet to induce HTN or maintained them on 0.4% NaCl normal salt (NS) diet and assessed mitochondrial bioenergetics and dynamics using spectrofluorimetry, Seahorse assay, electron paramagnetic resonance (EPR) spectroscopy, Western blotting, electron microscopy, PCR and cytokine assays. We report that under high salt conditions, associated with hypertension and renal damage, the SSNPPA-/- rats exhibit a decrease in mitochondrial membrane potential and elevation in mitochondrial ROS levels compared to SSWT. The redox shift is also evident by the presence of more pronounced medullar lipid peroxidation in the SSNPPA-/- strain. We also revealed fragmented, more damaged mitochondria in the SSNPPA-/- rats, accompanied by increased turnover and biogenesis. Overall, our data indicate that ANP deficiency causes disruptions in mitochondrial bioenergetics and dynamics which likely contributes to aggravation of the renal damage and hypertension in the Dahl SS rat; the major pathological effects are evident in the groups subjected to a combined salt and ANP deficiency-induced mitochondrial stress.

Spontaneous vascular dysfunction in Dahl salt-sensitive male rats raised without a high-salt diet.

Dahl salt-sensitive (SS) rats fed a high-salt diet, but not low-salt, exhibit vascular dysfunction. Several substrains of SS rats exist that differ in their blood pressure phenotypes and salt sensitivity. The goal of this study was to investigate whether the John-Rapp-derived SS rat (SS/Jr), which exhibits spontaneous hypertension on a low-salt diet, presents with hallmarks of vascular dysfunction observed in another experimental model of hypertension independent of dietary salt, the spontaneously hypertensive rat (SHR). Endothelium-intact aortic rings and mesenteric resistance arteries were isolated from low-salt fed adult male SS/Jr rats and SHRs, or their respective controls, for isometric wire myography. Vessels were challenged with cumulative concentrations of various vasoactive substances, in the absence or presence of nitric oxide synthase or cyclooxygenase inhibitors. Despite showing some differences in their responses to various vasoactive substances, both SS/Jr rats and SHRs exhibited key features of vascular dysfunction, including endothelial dysfunction and hyperresponsiveness to vasocontractile agonists. In conclusion, this study provides evidence to support the utility of the SS/Jr rat strain maintained on a low-salt diet as a valid experimental model for vascular dysfunction, a key feature of human hypertension.

Anti-atrial Fibrillatory and Cardiorenal Protective Effects of the Combination of Valsartan and Cilnidipine in Dahl Salt-Sensitive Rats.

The current study aimed to investigate the anti-atrial fibrillatory (AF) effects of a combination of valsartan and a calcium channel blocker (cilnidipine or amlodipine) in Dahl salt-sensitive (Dahl S) rats. Seven-week-old male Dahl S rats were fed an 8% salt diet. Six weeks later, valsartan (60 mg/kg, Val group), cilnidipine + valsartan (10 + 60 mg/kg, CV group), amlodipine + valsartan (3 + 60 mg/kg, AV group), or vehicle was orally administered daily for 5 weeks. Echocardiography and atrial electrophysiological evaluations were performed on the last day of treatment. Blood pressure in each drug treatment group was lower than in the Vehicle group. The duration of AF induced by atrial burst stimulation was shorter in the Val group (3.2 ± 1.6 s) than in the Vehicle group (11.2 ± 6.0 s), which was further shortened in the CV and AV groups (1.1 ± 0.3 and 1.3 ± 0.3 s, respectively). Left ventricular ejection fraction and left ventricular fractional shortening were greater in the CV and AV groups than those in the Vehicle group. Urinary albumin excretion in the CV group was the lowest among the drug-treated groups. The results collectively suggest that the combination of a calcium channel blocker with valsartan could be useful in terms of its anti-AF action as well as for improving cardiac and renal functions.

Integrins play a role in stress relaxation of perivascular adipose tissue.

Perivascular adipose tissue (PVAT) is known for being anti-contractile in healthy tissues. We discovered a new function of PVAT, the ability to stress relax and maintain a tone in response to a stretch. This is of note because stress relaxation has been attributed to smooth muscle, of which PVAT has none that is organized in a functional layer. We test the hypothesis the interactions of integrins with collagen play a role in stress relaxation. Our model is the thoracic aorta of the male Dahl SS rat. The PVAT and aorta were physically separated for most assays. Results from single nuclei RNA sequencing (snRNAseq) experiments, histochemistry and isometric contractility were also used. Masson Trichrome staining made evident the expression of collagen in PVAT. From snRNA seq experiments of the PVAT, mRNA for multiple collagen and integrin isoforms were detected: the α1 and ß1 integrin were most highly expressed. Pharmacological inhibition of integrin/collagen interaction was effected by the specific α1ß1 distintegrin obtustatin or general integrin inhibitor RGD peptide. RGD peptide but not obtustatin increased the stress relaxation. Cell-cell communication inference identified integrins αv and α5, two major RGD motif containing isoforms, as potential signaling partners of collagens. Collectively, these findings validate that stress relaxation can occur in a non-smooth muscle tissue, doing so in part through integrin-collagen interactions that may not include α1ß1 heterodimers. The importance of this lies in considering PVAT as a vascular layer that possesses mechanical functions.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Inhibition of mTORC2 promotes natriuresis in Dahl salt-sensitive rats via the decrease of NCC and ENaC activity.

We have previously observed that prolonged administration of rapamycin, an inhibitor targeting the mammalian target of rapamycin complex (mTORC)1, partially reduced hypertension and alleviated kidney inflammation in Dahl salt-sensitive (SS) rats. In contrast, treatment with PP242, an inhibitor affecting both mTORC1/mTORC2, not only completely prevented hypertension but also provided substantial protection against kidney injury. Notably, PP242 exhibited potent natriuretic effects that were not evident with rapamycin. The primary objective of this study was to pinpoint the specific tubular sites responsible for the natriuretic effect of PP242 in SS rats subjected to either 0.4% NaCl (normal salt) or 4.0% NaCl (high salt) diet. Acute effects of PP242 on natriuretic, diuretic, and kaliuretic responses were determined in unanesthetized SS rats utilizing benzamil, furosemide, or hydrochlorothiazide [inhibitors of epithelial Na+ channel (ENaC), Na-K-2Cl cotransporter (NKCC2), or Na-Cl cotransporter (NCC), respectively] either administered alone or in combination. The findings indicate that the natriuretic effects of PP242 in SS rats stem predominantly from the inhibition of NCC and a reduction of ENaC open probability. Molecular analysis revealed that mTORC2 regulates NCC activity through protein phosphorylation and ENaC activity through proteolytic cleavage in vivo. Evidence also indicated that PP242 also prevents the loss of K+ associated with the inhibition of NCC. These findings suggest that PP242 may represent an improved therapeutic approach for antihypertensive intervention, potentially controlling blood pressure and mitigating kidney injury in salt-sensitive human subjects.NEW & NOTEWORTHY This study explored mechanisms underlying the natriuretic effects of mammalian target of rapamycin protein complex 2 inhibition using PP242 and revealed both epithelial Na+ channel and Na-Cl cotransporter in the distal tubular segments were potentially inhibited. These observations, with prior lab evidence, indicate that PP242 prevents hypertension via its potent inhibitory effects on these specific sodium transporters and by reducing renal immune responses. This dual action, coupled with potassium sparing effects, suggests an improved approach for managing hypertension and associated kidney damage.

Sex differences in dietary sodium evoked NCC regulation and blood pressure in male and female Sprague-Dawley, Dahl salt-resistant, and Dahl salt-sensitive rats.

Hypertension affects approximately one in two United States adults and sex plays an important role in the pathogenesis of hypertension. The Na+-Cl- cotransporter (NCC), regulated by a kinase network including with-no-lysine kinase (WNK)1 and WNK4, STE20/SPS1-related proline alanine-rich kinase (SPAK), and oxidative stress response 1 (OxSR1), is critical to Na+ reabsorption and blood pressure regulation. Dietary salt differentially modulates NCC in salt-sensitive and salt-resistant rats, in part by modulation of WNK/SPAK/OxSR1 signaling. In this study, we tested the hypothesis that sex-dependent differences in NCC regulation contribute to the development of the salt sensitivity of blood pressure using male and female Sprague-Dawley (SD), Dahl salt-resistant (DSR), and Dahl salt-sensitive (DSS) rats. In normotensive salt-resistant SD and DSR rats, a high-salt diet evoked significant decreases in NCC activity, expression, and phosphorylation. In males, these changes were associated with no change in WNK1 expression, a decrease in WNK4 levels, and suppression of SPAK/OxSR1 expression and phosphorylation. In contrast, in females, there was decreased NCC activity associated with suppression of SPAK/OxSR1 expression and phosphorylation. In hypertensive DSS rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension. Collectively, our findings support the existence of sex differences in male versus female rats with NCC regulation during dietary salt intake involving suppression of WNK4 expression in male rats only and the involvement of SPAK/OxSR1 signaling in both males and females.NEW & NOTEWORTHY NCC regulation is sex dependent. In normotensive male and female Sprague-Dawley and Dahl salt-resistant rats, which exhibit dietary Na+-evoked NCC suppression, male rats exhibit decreased WNK4 expression and decreased SPAK and OxSR1 levels, whereas female rats only suppress SPAK and OxSR1. In hypertensive Dahl salt-sensitive rats, the ability of females to suppress NCC (in opposition to males) via a SPAK/OxSR1 mechanism likely contributes to their lower magnitude of salt-sensitive hypertension.

Effect of Low-Level Tragus Stimulation on Cardiac Metabolism in Heart Failure with Preserved Ejection Fraction: A Transcriptomics-Based Analysis.

Abnormal cardiac metabolism precedes and contributes to structural changes in heart failure. Low-level tragus stimulation (LLTS) can attenuate structural remodeling in heart failure with preserved ejection fraction (HFpEF). The role of LLTS on cardiac metabolism is not known. Dahl salt-sensitive rats of 7 weeks of age were randomized into three groups: low salt (0.3% NaCl) diet (control group; n = 6), high salt diet (8% NaCl) with either LLTS (active group; n = 8), or sham stimulation (sham group; n = 5). Both active and sham groups received the high salt diet for 10 weeks with active LLTS or sham stimulation (20 Hz, 2 mA, 0.2 ms) for 30 min daily for the last 4 weeks. At the endpoint, left ventricular tissue was used for RNA sequencing and transcriptomic analysis. The Ingenuity Pathway Analysis tool (IPA) was used to identify canonical metabolic pathways and upstream regulators. Principal component analysis demonstrated overlapping expression of important metabolic genes between the LLTS, and control groups compared to the sham group. Canonical metabolic pathway analysis showed downregulation of the oxidative phosphorylation (Z-score: -4.707, control vs. sham) in HFpEF and LLTS improved the oxidative phosphorylation (Z-score = -2.309, active vs. sham). HFpEF was associated with the abnormalities of metabolic upstream regulators, including PPARGC1α, insulin receptor signaling, PPARα, PPARδ, PPARGC1ß, the fatty acid transporter SLC27A2, and lysine-specific demethylase 5A (KDM5A). LLTS attenuated abnormal insulin receptor and KDM5A signaling. HFpEF is associated with abnormal cardiac metabolism. LLTS, by modulating the functioning of crucial upstream regulators, improves cardiac metabolism and mitochondrial oxidative phosphorylation.

Dissociation of Hypertension and Renal Damage After Cessation of High-Salt Diet in Dahl Rats.

BACKGROUND: Every year, thousands of patients with hypertension reduce salt consumption in an effort to control their blood pressure. However, hypertension has a self-sustaining character in a significant part of the population. We hypothesized that chronic hypertension leads to irreversible renal damage that remains after removing the trigger, causing an elevation of the initial blood pressure. METHODS: Dahl salt-sensitive rat model was used for chronic, continuous observation of blood pressure. Rats were fed a high salt diet to induce hypertension, and then the diet was switched back to normal sodium content. RESULTS: We found that developed hypertension was irreversible by salt cessation: after a short period of reduction, blood pressure grew even higher than in the high-salt phase. Notably, the self-sustaining phase of hypertension was sensitive to benzamil treatment due to sustaining epithelial sodium channel hyperactivity, as shown with patch-clamp analysis. Glomerular damage and proteinuria were also irreversible. In contrast, some mechanisms, contributing to the development of salt-sensitive hypertension, normalized after salt restriction. Thus, flow cytometry demonstrated that dietary salt reduction in hypertensive animals decreased the number of total CD45+, CD3+CD4+, and CD3+CD8+ cells in renal tissues. Also, we found tubular recovery and improvement of glomerular filtration rate in the postsalt period versus a high-salt diet. CONCLUSIONS: Based on earlier publications and current data, poor response to salt restriction is due to the differential contribution of the factors recognized in the developmental phase of hypertension. We suggest that proteinuria or electrolyte transport can be prioritized over therapeutic targets of inflammatory response.

Abatacept Decreases Renal T-cell Infiltration and Renal Inflammation and Ameliorates Progressive Renal Injury in Obese Dahl Salt-sensitive Rats Before Puberty.

ABSTRACT: Prepubertal obesity is growing at an alarming rate and is now considered a risk factor for renal injury. Recently, we reported that the early development of renal injury in obese Dahl salt-sensitive (SS) leptin receptor mutant (SS LepR mutant) rats was associated with increased T-cell infiltration and activation before puberty. Therefore, the current study investigated the effect of inhibiting T-cell activation with abatacept on the progression of renal injury in young obese SS LepR mutant rats before puberty. Four-week-old SS and SS LepR mutant rats were treated with IgG or abatacept (1 mg/kg; ip, every other day) for 4 weeks. Abatacept reduced the renal infiltration of T cells by almost 50% in SS LepR mutant rats. Treatment with abatacept decreased the renal expression of macrophage inflammatory protein-3 alpha while increasing IL-4 in SS LepR mutant rats without affecting SS rats. While not having an impact on blood glucose levels, abatacept reduced hyperinsulinemia and plasma triglycerides in SS LepR mutant rats without affecting SS rats. We did not observe any differences in the mean arterial pressure among the groups. Proteinuria was markedly higher in SS LepR mutant rats than in SS rats throughout the study, and treatment with abatacept decreased proteinuria by about 40% in SS LepR mutant rats without affecting SS rats. We observed significant increases in glomerular and tubular injury and renal fibrosis in SS LepR mutant rats versus SS rats, and chronic treatment with abatacept significantly reduced these renal abnormalities in SS LepR mutant rats. These data suggest that renal T-cell activation contributes to the early progression of renal injury associated with prepubertal obesity.

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.