DOCA-salt Model Research Articles

Role of Urokinase in Chronic Hypertension with Albuminuria in DOCA-Salt Murine Model

Role of Urokinase in Chronic Hypertension with Albuminuria in DOCA-Salt Murine Model

Abstract P200: Immune memory contributes to chronic hypertension recurrence

Keeping blood pressure under control is a challenge in hypertension treatment because high blood pressure quickly returns with a momentary lapse in treatment or a high-salt diet. We have previously reported the CD8+ T cells (CD8Ts) in the kidney can drive aberrant sodium handling to aggravate blood pressure. Recently, our new findings suggest that these CD8Ts remain long-term within the kidney. Here, we propose these CD8Ts have developed into a resident memory (Trm) population with the aid of TGFβ signaling, and this residency continually aggravates and leads to the recurrence of hypertension . To explore this hypothesis, we utilized two mouse models of hypertension, including a classic DOCA-salt model and a novel Angiotensin II (Ang II) induced salt memory model, with continuous blood pressure monitored by radiotelemetry. In the Ang II-induced salt memory model, the mice were administered Ang II and high salt for a week before a resting period to restore normal blood pressure. Thereafter, the mice were given a second high salt challenge to model the high salt-driven recurrence of hypertension. Mouse strains include both wild-type (WT) and a T-cell specific TGFβRII KO (Tsp-TGFβR KO), which has T cells unable to develop into Trm due to the reliance of Trm on TGFβ signaling. T cells were analyzed with flow cytometry. In the DOCA-salt model, WT mice developed a significant CD8Trm population within the kidney. In contrast, TSP-TGFβR KO mice, which lack the ability to develop Trm, had an attenuated development of hypertension with DOCA-salt treatment. Testing the capability for hypertension to recur, both WT and Tsp-TGFβR KO mice experienced a similar degree of hypertension with Ang II-salt treatment. However, only the WT had a significant expansion of Trm and demonstrated the clinical manifestation of recurring hypertension with the rapid increase of blood pressure with a high salt challenge. Isolating the T cells from the kidneys confirmed that the significant CD8Trm population development in the WT was not present in the Tsp-TGFβR KO. In summary, our findings suggest that the memory T cell population developed in the kidney during hypertension instills a perpetual memory for salt sensitivity in hypertension. Without the ability to develop this memory population, mice are protected against this recurrence of high-salt-induced hypertension.

O14 KIDNEY RESIDENT MEMORY T CELLS MEDIATE THE CHRONIC PROGRESSION OF HYPERTENSION

A significant challenge in treating hypertension is insufficient blood pressure (BP) control and its recurrence. Previously, we described the contribution of CD8 T cells (CD8Ts) to the pathogenesis of salt-sensitive hypertension. Notably, despite antihypertensive therapy temporarily lowering BP in hypertensive mice, the T cell residency was not disrupted within the kidneys, and hypertension recurred when treatment was ceased. We hypothesize that the CD8Ts established a long-living resident memory (Trm) population in the kidney that instigates the recurrence of hypertension. To examine this, we utilized both wild-type (WT) and T-cell-specific KO of TGFβRII (Tsp-TGFβRKO) mice, which prevents the formation of Trms. Blood pressure was recorded continuously with radiotelemetry. Hypertension was induced with the classic DOCA-salt model and an Angiotensin II (Ang II)-induced salt memory model to mimic the clinical recurrence of hypertension with high salt intake. In this salt-memory model, a one-week infusion of Ang II paired with high salt-induced hypertension before being allowed to return to normal blood pressure. A chronic high salt intake challenge then tested the recurrence of hypertension. Both splenic and renal T cell populations were characterized using flow cytometry. As expected, DOCA-salt treatment led to salt-sensitive hypertension in WT mice along with the development of a significant CD8Trm population within their kidneys. These CD8Trms were notably diminished in the kidneys of DOCA-treated Tsp-TGFβRKO mice who also had attenuated blood pressure elevation. While testing the recurrence of hypertension during the Ang II-induced salt-memory model both WT and Tsp-TGFβKO reached the same degree of hypertension during Ang II-salt and baseline blood pressure before and after Ang II-salt. However, only the WT developed Trm and had the recurrence of hypertension with high salt challenge whereas both were lost in the Tsp-TGFβKO mice. In summary, our study unveils the critical role of kidney CD8Trms in contributing to salt-sensitive hypertension and its recurrence. This leads to targeting key molecules that initiate the development of Trms to mitigate the persistency of hypertension.

Antidyslipidaemic evaluation of coagulin C and withacoagulin isolated from Withania coagulans (Stocks) dunal berries in hypertension induced secondary dyslipidemia in albino mice

Vascular diseases like hypertension has often been associated with dyslipidemia. Sustained elevated blood pressure for not less than four weeks develops gross irregularities inside arteriolar lumen. The pathological changes might be due to intimal arteriosclerotic thickening causing tapering of the structural narrowing along the arterioles. It might also be due to occurrence of irregular damage in the vascular structure from focal arteriolar constriction and lead to secondary dyslipidemia. The pharmacological intervention may lead to rectification and therapeutic benefit against dyslipidemia.The withanolides coagulin C and withacoagulin were isolated by flash chromatography from the extracts of Withania coagulans (Stocks) Dunal berries. The withanolides were characterized using 1H NMR and 13C NMR, UV and mass spectrometry and confirmed as coagulin C and withacoagulin. The oral toxicity of coagulin C and withacoagulin were investigated in albino mice followed by their antidyslipidemic activity with two dose levels i.e. 25 mg/kg and 50 mg/Kg p.o. This dyslipidemia was secondary to hypertension which was developed by DOCA salt model (25 mg/kg; s.c.+ 2% NaCl). Mice were administered dose of 10, 50 and 500 mg/kg coagulin C and withacoagulin orally for 28 days daily for sub-acute toxicity effect.Coagulin C and withacoagulin were found to be tolerated upto 2000 mg/kg p.o. during acute toxicity study in mice. Coagulin C and withacoagulin were claimed to possess No-Observed Adverse Effect Level (NOEL) at the dose of 500 mg/kg orally. The high dose (50 mg/Kg) withacoagulin has highly significant difference in total cholesterol, triglyceride, HDL, LDL and VLDL levels in comparison to disease control group along with highly significant difference in triglyceride and VLDL level compared to animals of vehicle control group. Coagulin C at both the low (25 mg/Kg) and high dose (50 mg/Kg) have highly significant difference in comparison to vehicle control, disease control and positive control in controlling total cholesterol, triglyceride, LDL and VLDL levels in the experimental animals indicating that coagulin C is not much as effective as withacoagulin in the normalization of altered lipid profile of the experimental animals.On the basis of spectroscopic analysis the isolated compounds from Withania coagulans (Stocks) Dunal berries were confirmed to be coagulin C and withacoagulin. Acute oral toxicity test in mice was found to have safe profile even at high dose for both the compounds. On development of secondary dyslipidemia due to DOCA salt induced hypertension in albino mice, both the isolated compounds showed different degree of correction in the altered lipid profile of the animals. Whereas withacoagulin has been found to have more significant and successful in controlling the altered lipid profile, coagulin C has only marginal effect. The docking studies also confirm the result and hence it can be suggested that withacoagulin is better than coagulin C in managing the alteration of lipid profile of the experimental mice.

Sex Dependent Exacerbation of Salt Sensitive Hypertension and Cardiac Dysfunction Is Associated With Upregulation of miRNA Targeting ADAM17 and ACE2 in the Brain

Background: The DOCA-salt model has been widely used to study salt-sensitive hypertension and we previously reported that it is associated with ADAM17-mediated ACE2 shedding in the central nervous system. However, previously reported data are mainly from male mice, and it is unclear whether female mice exhibit the same phenotypes. Objective: We aimed to compare sex specific development of salt sensitive hypertension and cardiac dysfunction and investigate miRNA regulation associated with these changes. Methods: C57 mice (male and females, n = 8 -15) underwent uninephrectomy and telemetry implantation (DSI Inc.) for blood pressure (BP) conscious recording. After baseline recording, a DOCA pellet (50 mg, 21 days, Innovative Research of America) was implanted subcutaneously and drinking water was replaced by 1% NaCl solution to induce salt sensitive hypertension. Cerebral Spinal fluid (CSF) was collected at baseline and after 3 weeks of DOCA-salt treatment for exosome miRNA sequencing (Creative Biolab). In another set of mice, cardiac function was assessed by echocardiography (Visualsonics Vevo F2) and invasive left ventricular hemodynamics (Transonic). Mortality from each group was analyzed as well. Results: Baseline BP was not different between male and female mice. Following DOCA-salt treatment for 3 weeks, BP was increased in both sexes, with higher systolic BP observed in males compared to females (active phase: 173.0 ± 19.6 vs. 154.2 ± 19.1 mmHg, p<0.05). Although ejection fraction was not changed in either male or female mice, mitral peak early diastolic filling velocity and tissue doppler showed that E/E’ was increased in both sexes compared to baselines (Male: 33.4 ± 4 vs. 26.6 ± 1.9; Female: 30.3 ± 5.0 vs. 26.4 ± 4), with a greater increase in males (25.3% vs. 14.4%). In addition, hemodynamics revealed increased left ventricular end-diastolic pressure (11.1 ± 1.9 vs. 4.5 ± 0.5 mmHg, p<0.01) and relaxation time constant Tau (6.7 ± 1.0 vs. 4.9 ± 0.5 msec, p<0.01) in DOCA-salt treated males compared to non-treated controls, with no changes shown in female mice. These data suggest worsened cardiac diastolic function in males compared to females following DOCA-salt treatment. Moreover, a 20% mortality was observed in males while no death occured in females, confirming a more severe phenotype in males. Furthermore, miRNAseq analysis of CSF exosomes showed several upregulated miRNA in males but not in females following DOCA-salt treatment. Notably, miR206 associated with increased ADAM17 activity was increased by 8-fold in males but only 3-fold n females. Conclusion: DOCA-salt treatment promotes exacerbated hypertension, impaired cardiac function and mortality in a sex dependent manner, associated with upregulation of key miRNA targeting the brain renin-angiotensin system. The changes of ADAM17 and ACE2 targeted exosome miRNAs in the central nervous system might contribute to the sex difference in the cardiovascular dysfunction phenotypes in this model. This work was supported in part by a research grant from the National Institutes of Health (HL163588). This is the full abstract presented at the American Physiology Summit 2024 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.

Egg white hydrolysate protects white adipose tissue against metabolic insult in deoxycorticosterone acetate-salt rats.

The purpose of this study was to investigate the effect of an egg white hydrolysate (EWH) to protect white adipose tissue damage from cardiometabolic changes induced by severe hypertension. Male Wistar rats were uninephrectomised and divided: SHAM (weekly subcutaneous vehicle (mineral oil + propylene glycol, 1:1)), SHAM + EWH (subcutaneous vehicle plus EWH via gavage, 1 g/kg per day), DOCA (deoxycorticosterone acetate diluted in vehicle subcutaneously weekly in subsequent doses of 20 mg/kg -1st week, 12 mg/kg - 2–3th week, and 6 mg/kg -4–8th week, respectively, plus 1 % NaCl and 0·2 % KCl in drinking water), and DOCA + EWH. Body weight gain, food and water intake, glucose and lipid metabolism were evaluated. Oxidative stress was assessed by biochemical assay and immunofluorescence for NOX-1, nuclear factor kappa B (NFκB), and caspase-3 in retroperitoneal white adipose tissue (rtWAT). Proinflammatory cytokines (IL-6 and 1β), CD163+ macrophage infiltration, and immunohistochemistry for TNFα and uncoupling protein-1 were evaluated, as well as histological analysis on rtWAT. Glutathione peroxidase and reductase were also determined in plasma. EWH showed hypocholesterolemic, antioxidant, anti-inflammatory, and anti-apoptotic properties in the arterial hypertension DOCA-salt model. The results demonstrated the presence of functional changes in adipose tissue function by a decrease in macrophage infiltration and in the fluorescence intensity of NFκB, NOX-1, and caspase-3. A reduction of proinflammatory cytokines and restoration of antioxidant enzymatic activity and mitochondrial oxidative damage by reducing uncoupling protein-1 fluorescence intensity were also observed. EWH could be used as a potential alternative therapeutic strategy in the treatment of cardiometabolic complications associated with malignant secondary arterial hypertension.

Abstract 15056: The Activation of Angiotensin II Type1a Receptors in the SFO and PVN is Necessary to Prime Immunity in Salt Sensitive Hypertension

Introduction: Salt sensitive hypertension (HTN) is associated with increased salt intake and elevated activity of the renin-angiotensin system (RAS) in the brain. We have found that DOCA-salt HTN partly depends on a sympathetic-mediated PlGF release in the spleen. Hypothesis: Whether this effect is related to the contribution of RAS signaling in brain nuclei mediating HTN responses is poorly known. We hypothesized that angiotensin type 1a receptors (AT1aR) within the circumventricular organs (CVOs) could mediate DOCA-salt hypertensive responses. Methods: To discriminate between CVOs, we used 3 mouse model with deletion of AT1aR in 3 in brain CVOs. AT1aRfloxed mice were 1) stereotaxically injected with an adenovirus encoding a Cre-recombinase (or GFP as control), directly in the subfornical organ (SFO), or 2) crossed with Sim1Cre+ (and Sim1Cre- as control) mice to delete the gene in the paraventricular nucleus of hypothalamus (PVN) or 3) crossed with Phox2bCre+ (and Phox2bCre- as control) mice to delete the gene in the dorsal motor nucleus of the vagus nerve (DMV). T Results: We implanted mice with pellets of DOCA or placebo, as control, and salt in drinking water for 3 weeks finding that: AT1aRfloxed mice + Virus encoding Cre Recombinase in the SFO and AT1aRfloxed;Sim1Cre+ mice were protected form HTN-DOCA-salt (SFO-SBP mmHg :133±1 vs 106±0,6, ***p>0,0001; PVN-SBP mmHg :144±2 vs 112±2,***p>0,0001 respectively to their controls); whereas AT1aRfloxed;Phox2bCre+ mice had increased blood pressure levels (SBP mmHg :144±1 vs 144±0,7, respectively to control mice). Additionally, deletion of AT1aR in the SFO and PVN attenuated egression of T cells from the spleen and infiltration on target organ with a reduced disruption of renal glomeruli and reduced accumulation of matrix protein in response to DOCA-salt (SFO-Bowman’s size μm 2 1761±150 vs 821±78, respectively, **p<0,001; PVN-Bowman’s size μm 2 3927±212 vs 2805±252, respectively, **p<0,001). Mice KO for AT1aR in the DMV were not protected from organ damage (Bowman’s size μm 2 776 ± 104 vs 1090 ± 94, respectively). Conclusions: ogether, these data indicate that SFO and PVN contribute to blood pressure in the DOCA-salt model of hypertension through AT1aR signaling, while the DMV role was independent from AT1aR.

P2Y2 receptor decreases blood pressure by inhibiting ENaC.

Stimulating the Gq-coupled P2Y2 receptor (P2ry2) lowers blood pressure. Global knockout of P2ry2 increases blood pressure. Vascular and renal mechanisms are believed to participate in P2ry2 effects on blood pressure. To isolate the role of the kidneys in P2ry2 effects on blood pressure and to reveal the molecular and cellular mechanisms of this action, we test here the necessity of the P2ry2 and the sufficiency of Gq-dependent signaling in renal principal cells to the regulation of the epithelial Na+ channel (ENaC), sodium excretion, and blood pressure. Activating P2ry2 in littermate controls but not principal cell-specific P2ry2-knockout mice decreased the activity of ENaC in renal tubules. Moreover, deletion of P2ry2 in principal cells abolished increases in sodium excretion in response to stimulation of P2ry2 and compromised the normal ability to excrete a sodium load. Consequently, principal cell-specific knockout of P2ry2 prevented decreases in blood pressure in response to P2ry2 stimulation in the deoxycorticosterone acetate-salt (DOCA-salt) model of hypertension. In wild-type littermate controls, such stimulation decreased blood pressure in this model of hypertension by promoting a natriuresis. Pharmacogenetic activation of Gq exclusively in principal cells using targeted expression of Gq-designer receptors exclusively activated by designer drugs and clozapine N-oxide decreased the activity of ENaC in renal tubules, promoting a natriuresis that lowered elevated blood pressure in the DOCA-salt model of hypertension. These findings demonstrate that the kidneys play a major role in decreasing blood pressure in response to P2ry2 activation and that inhibition of ENaC activity in response to P2ry2-mediated Gq signaling lowered blood pressure by increasing renal sodium excretion.

Immune profiling of murine cardiac leukocytes identifies triggering receptor expressed on myeloid cells 2 as a novel mediator of hypertensive heart failure.

Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction, microvascular dysfunction, and myocardial fibrosis with recent evidence implicating the immune system in orchestrating cardiac remodelling. Here, we show the mouse model of deoxycorticosterone acetate (DOCA)-salt hypertension induces key elements of HFpEF, including diastolic dysfunction, exercise intolerance, and pulmonary congestion in the setting of preserved ejection fraction. A modified single-cell sequencing approach, cellular indexing of transcriptomes and epitopes by sequencing, of cardiac immune cells reveals an altered abundance and transcriptional signature in multiple cell types, most notably cardiac macrophages. The DOCA-salt model results in differential expression of several known and novel genes in cardiac macrophages, including up-regulation of Trem2, which has been recently implicated in obesity and atherosclerosis. The role of Trem2 in hypertensive heart failure, however, is unknown. We found that mice with genetic deletion of Trem2 exhibit increased cardiac hypertrophy, diastolic dysfunction, renal injury, and decreased cardiac capillary density after DOCA-salt treatment compared to wild-type controls. Moreover, Trem2-deficient macrophages have impaired expression of pro-angiogenic gene programmes and increased expression of pro-inflammatory cytokines. Furthermore, we found that plasma levels of soluble TREM2 are elevated in DOCA-salt treated mice and humans with heart failure. Together, our data provide an atlas of immunological alterations that can lead to improved diagnostic and therapeutic strategies for HFpEF. We provide our dataset in an easy to explore and freely accessible web application making it a useful resource for the community. Finally, our results suggest a novel cardioprotective role for Trem2 in hypertensive heart failure.

Macrophage to Myofibroblast Transition in Diastolic Dysfunction Development

Heart failure is the top killer in the U.S and worldwide, often caused by hypertension, aging, or cardiac toxicity. Diastolic Dysfunction (DD), the often the first step of heart failure, occurs when the chambers of the heart are not able to expand during the filling phase. In addition, hypertension is the leading cause of DD. Though this is known, the progression of hypertension into DD remains poorly understood. Of note is the ability of M2 macrophages to differentiate into a myofibroblast-like phenotype, known as macrophage to myofibroblast transition (MMT). This transition has been observed in interstitial fibrosis in chronic renal allograft injury. The researchers noted that MMT account for 50% of the myofibroblast population. To our knowledge, the extent of fibrotic remodeling due to MMT in the heart has not been ardently investigated. Therefore, we aim to answer whether MMT plays a causal role in the progression of hypertension to DD and the pathogenesis of heart failure.Utilizing the uninephrectomized, DOCA-salt model, mice were subjected to this treatment and hypertension was induced. As a negative control, another group of mice received a uninephrectomy and the same salt in their drinking water, however, DOCA was not administered (Sham group). MMT prevalence, identified as CD68+ α-SMA+ expressions, was observed inside the hearts of DOCA and Sham mice. The hearts were isolated and homogenized via gentleMACS tissue Dissociator, the monocytes were purified via Percoll isolation, and flow cytometry was used to identify the population of CD68+ α-SMA+ expressing cells.As soon as 10 days post DOCA implantation, the DOCA group exhibited worsening diastolic function as it took longer for the pressure to change in the hearts of DOCA mice than in Sham (-dp/dt). These data were gathered via left ventricular (LV) catheter insertion. At a later time, utilizing a different cohort of mice preliminary data show an increase of monocyte (CD68+) and fibroblast (α-SMA+) double-positive expressing cells in the hearts of hypertensive mice.MMT has been observed in the hearts of hypertensive mice. This transition may exacerbate Diastolic Dysfunction. More research must be performed to investigate the wide variety of functions of macrophages (i.e., M1 to M2 ratio and transition, macrophage residence or infiltration, and macrophage stimulation of myofibroblasts), as well as the specific interactions between macrophages and cardiomyocytes, or other immune cells, that might mediate this transition. These data provide a preliminary, but interesting glimpse, into the role of macrophages in exacerbating Diastolic Dysfunction and mediating Heart Failure. NIH RO1-HL146713 and AHA 15BGIA25730047 (Shengyu Mu) 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.

S-40-5: ROLE OF INTERFERON GAMMA IN T CELL MEDIATED SALT-RETENTION IN THE KIDNEY AND PROGRESSION OF HYPERTENSION

Objective: In accordance with previous studies implicating the immune system in the development of hypertension, we found that CD8+ T cells (CD8Ts) infiltrate the kidneys and stimulate distal convoluted tubular cells (DCTs), to upregulate sodium-chloride co-transporter (NCC) activity, leading to excessive salt retention. However, the precise molecules driving this direct interaction between CD8Ts and DCTs have not yet been identified. Here we hypothesize that gamma interferon (IFNg), released by activated CD8Ts, primes the tubular cell to promote interactions between activated CD8Ts and DCTs during the development of salt-sensitive hypertension. We predict blocking this molecular pathway will reduce CD8T-homing into the kidney, lowering blood pressure. Design & Method: In co-culture of mouse CD8Ts and DCTs, recombinant mIFNg, IFNg-neutralizing antibody, and specific siRNAs against the IFNg-receptor or PDL1 were used to determine the role of IFNg-PDL1 pathway in the CD8T-DCT interaction. To validate these studies, we employed IFNg knockout (KO) mice, mice with systemically blocked PDL1 using anti-PDL1 antibody or with renal tubule-specific knockdown of PDL1 (mediated via nanoparticles) in the DOCA-salt model and the CD8T adoptive transfer hypertension models. Blood pressure was monitored using radio-biotelemetry. Endpoint analysis involved flow cytometry, RT-qPCR, and western blot, and immunohistochemistry. Results: We found that CD8Ts isolated from DOCA-salt treated hypertensive mice produce more IFNg and exhibit augmented ability to interact with DCTs compared to those from sham normotensive mice. IFNg stimulation to mDCTs increased expression of CD8-specific antigen presenting molecule MHC-I and CD8T-co-signaling molecule PDL1, which enhanced the interaction between CD8Ts and DCTs, consequently upregulating NCC expression in DCTs, leading to enhanced salt retention. These effects on MHC-I, PDL1, or NCC expression were abolished by neutralizing IFNg in co-culture of DCTs and activated CD8Ts, and knockdown of PDL1 in DCTs blunted NCC expression and sodium retention. In-vivo results verified the in-vitro studies. We found that in both IFNg-KO mice and WT mice receiving PDL1-antagonizing antibodies, DOCA-salt treatment failed to elevate their blood pressure and reduced T cell infiltration and NCC expression within their kidneys compared to WT mice receiving the same treatment (anti-PDL1 results shown in Figure 1). Similar results were observed in DOCA-salt treated mice with renal tubule-specific knockdown of PDL1. Conclusion: In salt-sensitive hypertension, activated CD8Ts demonstrate enhanced ability to interact with DCTs leading to increased expression of NCC and sodium retention through a IFNg-PDL1 pathway mediated mechanism. Blocking the IFNg-PDL1 pathway prevents CD8T-DCT interaction both in vitro and in vivo, ameliorating hypertension.

S-08-3: ROCK2 SPECIFIC INHIBITION DECREASES TH17/TREG BALANCE AND REDUCES HYPERTENSION AND HYPERTENSION-ASSOCIATED END-ORGAN DAMAGE

Hypertension is characterized by immune activation and inflammatory end-organ damage, yet there are no therapies for hypertension that specifically target the immune system. We previously demonstrated a critical role for pro-inflammatory CD4+ T cells and their cytokines, interleukin 17A (IL-17A) and IL-21, in the full development of hypertension and hypertension-associated renal and vascular damage. Rho-associated coiled-coil containing protein kinase 2 (ROCK2) has been shown to function as a molecular switch upregulating Th17 and inhibiting anti-inflammatory regulatory T cell (Treg) differentiation in autoimmune diseases. We found that ROCK2 expression is upregulated in splenic and renal CD4+ T cells in a DOCA (deoxycorticosterone) salt model of hypertension. We hypothesized that hypertensive stimuli increase T cell ROCK2 activity, leading to increased Th17/Treg ratios, and that selective ROCK2 inhibition is a potential novel therapeutic target for hypertension-associated end-organ damage. We showed in vitro that KD025, an orally bioavailable ROCK2 inhibitor, inhibits Th17 proliferation and IL-17A/IL-21 production. We then tested ROCK2 inhibition in vivo where DOCA salt-induced hypertension (uninephrectomy + 100 mg DOCA pellet + 1% NaCl drinking water) was initiated in all mice allowing for hypertension to develop for 10 days, followed by treatment with KD025 (50 mg/kg daily, n = 11) or vehicle (n = 8) starting on day 11 through the remainder of the protocol. KD025 treatment significantly attenuated cardiac hypertrophy (8.7 ± 0.3 vs 7.6 ± 0.2 mg/grams body weight, p = 0.005) and left ventricular (LV) fibrosis (4.9 ± 1.7 vs 2.4 ± 1.3% LV area, p = 0.008). Flow cytometric analysis revealed that KD025 decreased F4/80+ infiltrating cardiac macrophages (24,000 ± 578 vs 18,918 ± 1,430 cells, p = 0.011) and decreased the Th17/Treg ratio (0.251 ± 0.041 vs 0.117 ± 0.009, p = 0.003). Furthermore, KD025 attenuated renal infiltration of CD3+ T cells (59.0 ± 5.6 vs 43.5 ± 3.8 cells/mg tissue, p = 0.019). Additional studies using the Angiotensin II (Ang II, 490ng/kg/min) model of hypertension demonstrated that KD025 treatment starting on Day 14 of Ang II infusion significantly reduced blood pressures from Day 14 (154 ± 5.5mmHg) to Day 28 of the study (141.9 ± 5.9mmHg, p = 0.043, n = 5) which was not observed in vehicle controls (Day 14 157.0 ± 5.4mmHg, Day 28 154.9 ± 3.7mmHg, n = 4). These data indicate that the ROCK2-specific inhibitor KD025 may be a novel therapeutic for the treatment of hypertension and the associated end-organ damage.

S-12-4: CITE-SEQ IDENTIFIES TREM2-DEPENDENT GENE PROGRAM DRIVING CARDIAC REMODELING IN A HYPERTENSIVE MODEL OF HEART FAILURE WITH PRESERVED EJECTION FRACTION

Objective: The deoxycorticosterone acetate (DOCA)-salt model of hypertension has been used to study diastolic dysfunction and heart failure with preserved ejection fraction (HFpEF) in rats. Our goal was to validate DOCA-salt as a mouse model of HFpEF and to identify immune mediators of cardiac remodeling using CITE-Seq, a novel single cell technique that provides surface marker and transcriptomic data. Methods: DOCA-salt mice underwent uninephrectomy, DOCA pellet implantation, and 1% NaCl supplementation of the drinking water for three weeks. Control mice underwent a sham procedure and received normal water. Echocardiography and invasive hemodynamics were performed to examine cardiac function, and urine was collected to examine renal function. CITE-seq was performed on sorted leukocytes from four sham and DOCA-salt left ventricles. To test whether Trem2 has a causal role in HFpEF, wild type and Trem2 deficient animals were subjected to DOCA-salt. To evaluate translational potential, soluble Trem2 was measured in plasma of patients with HFpEF and healthy controls. Results: Compared to control mice, DOCA-salt mice exhibit significantly increased systolic blood pressure, albuminuria, and cardiac hypertrophy. On echocardiography, DOCA-salt mice exhibited a preserved ejection fraction. Invasive hemodynamic studies revealed an increased tau constant (5.70 ± .24, 8.22 ± .45; n = 8,5) in DOCA-salt mice, indicating diastolic dysfunction. CITE-seq revealed major transcriptional changes in cardiac macrophages between groups with upregulation of genes known to be involved in HFpEF and cardiac remodeling, such as galectin-3 (Lgals3) and osteopontin (Spp1), as well as novel genes including Trem2. Using an independent cohort of Sham and DOCA-salt treated mice, we validated a two-fold increase in Trem2 gene expression in left ventricles by qRT-PCR and a significant increase in Trem2 mRNA in Cd68+ macrophages by RNAscope. After DOCA-salt treatment, Trem2-deficient mice displayed increased heart weight to tibia length ratios (9.39 ± 0.20, 11.21 ± 0.80; n = 6–7) and albumin to creatinine ratios (5.5 ± 1.2, 14.2 ± 2.9; n = 6–7) compared to wild-type controls. No difference in albuminuria was observed at baseline (0.36 ± 0.05, 0.46 ± 0.10; n = 10). Bone marrow-derived macrophages (BMDMs) from Trem2 deficient mice expressed significantly less Lgals3 and Spp1 compared to wild type BMDMs suggesting that Trem2 signaling is upstream of galectin 3 and osteopontin in HFpEF. Plasma levels of soluble Trem2 were elevated in patients with HFpEF compared to healthy controls (51 ± 9.5; 22.4 ± 2.4 ng/mL; n = 9,12). Conclusions: The DOCA-salt mouse model induces key features of human HFpEF and identifies a Trem2-dependent gene program involved in cardiac remodeling. Loss of Trem2 resulted in increased cardiac hypertrophy and renal damage in the DOCA-salt model of HFpEF.

The DOCA-Salt Model of Hypertension for Studies of Cerebrovascular Function, Stroke, and Brain Health.

The brain renin-angiotensin-aldosterone system (RAAS) regulates many physiological processes including fluid and electrolyte balance, vascular structure and function, blood pressure, cognition, and other aspects of brain function. Treatment with the mineralocorticoid deoxycorticosterone acetate and salt stimulates the local RAAS within the brain. In this chapter, we describe the surgical procedures used to induce activation of the brain RAAS with deoxycorticosterone acetate and salt. This technique can be used for studies of hypertension, cerebrovascular biology and dysfunction, and other diseases that impact brain health.

ROS Suppression by Egg White Hydrolysate in DOCA-Salt Rats—An Alternative Tool against Vascular Dysfunction in Severe Hypertension

This study aimed to evaluate the potential for lowering blood pressure and beneficial effects on mesenteric resistance arteries (MRA) and conductance vessels (aorta) produced by dietary supplementation of an egg white hydrolysate (EWH) in rats with severe hypertension induced by deoxycorticosterone plus salt treatment (DOCA-salt), as well as the underlying mechanisms involved. The DOCA-salt model presented higher blood pressure, which was significantly reduced by EWH. The impaired acetylcholine-induced relaxation and eNOS expression observed in MRA and aorta from DOCA-salt rats was ameliorated by EWH. This effect on vessels (MRA and aorta) was related to the antioxidant effect of EWH, since hydrolysate intake prevented the NF-κB/TNFα inflammatory pathway and NADPH oxidase-induced reactive oxygen species (ROS) generation, as well as the mitochondrial source of ROS in MRA. At the plasma level, EWH blocked the higher ROS and MDA generation by DOCA-salt treatment, without altering the antioxidant marker. In conclusion, EWH demonstrated an antihypertensive effect in a model of severe hypertension. This effect could be related to its endothelium-dependent vasodilator properties mediated by an ameliorated vessel’s redox imbalance and inflammatory state.

Finerenone Reduces Renal RORγt γδ T Cells and Protects against Cardiorenal Damage

Introduction: Chronic activation of the mineralocorticoid receptor (MR) leads to pathological processes like inflammation and fibrosis during cardiorenal disease. Modulation of immunological processes in the heart or kidney may serve as a mechanistic and therapeutic interface in cardiorenal pathologies. In this study, we investigated anti-inflammatory/-fibrotic and immunological effects of the selective nonsteroidal MR antagonists finerenone (FIN) in the deoxycorticosterone acetate (DOCA)-salt model. Methods: Male C57BL6/J mice were uninephrectomized and received a DOCA pellet implantation (2.4 mg/day) plus 0.9% NaCl in drinking water (DOCA-salt) or received a sham operation and were orally treated with FIN (10 mg/kg/day) or vehicle in a preventive study design. Five weeks after the procedure, blood pressure (BP), urinary albumin/creatinine ratio (UACR), glomerular and tubulointerstitial damage, echocardiographic cardiac function, as well as cardiac/renal inflammatory cell content by FACS analysis were assessed. Results: BP was significantly reduced by FIN. FACS analysis revealed a notable immune response due to DOCA-salt exposure. Especially, infiltrating renal RORγt γδ-positive T cells were upregulated, which was significantly ameliorated by FIN treatment. This was accompanied by a significant reduction of UACR in FIN-treated mice. In the heart, FIN reduced DOCA-salt-induced cardiac hypertrophy, cardiac fibrosis and led to an improvement of the global longitudinal strain. Cardiac actions of FIN were not associated with a regulation of cardiac RORγt γδ-positive T cells. Discussion/Conclusion: The present study shows cardiac and renal protective effects of FIN in a DOCA-salt model. The cardiorenal protection was accompanied by a reduction of renal RORγt γδ T cells. The observed actions of FIN may provide a potential mechanism of its efficacy recently observed in clinical trials.

Investigation of selective vagal afferents subserving baroreflex function and blood pressure control: implications for understanding and alleviating hypertension

Cardiovascular homeostasis is maintained, in part, by neural signals arising from arterial baroreceptors (BRs) that apprise the brain of blood volume and pressure within the carotid sinus and aortic arch. Here, we test the overall hypothesis that neurons within the nodose ganglia that express the angiotensin type 1a receptor (referred to as NDGAT1aR) serve as BR that undergo plasticity during chronic elevations in blood pressure (BP). Towards this end, mice that express Cre‐recombinase at the AT1aR locus were delivered a Cre‐dependent AAV to express tdTomato within NDGAT1aR. These studies revealed that NDGAT1aR receive input from the aortic arch and carotid sinus, project to the caudal nucleus of the solitary tract (cNTS), and synthesize mechanosensitive ion channels, Piezo‐1/‐2. To determine whether NDGAT1aR couple their firing to elevated BP, we generated mice with the expression of the fluorescent Ca2+ indicator, GCaMP6s, directed to the AT1aR locus. Two‐photon (2P) intravital imaging revealed that phenylephrine (PE; i.v.) significantly increased GCaMP6s fluorescence within NDGAT1aR. To determine if optogenetic perturbation of NDGAT1aR at their soma or axon terminals within the cNTS affect the baroreflex, we directed the expression of excitatory (channelrhodpsin2; ChR2) or inhibitory (halorhodopsin; Halo) opsins to the AT1aR locus. Optical excitation of NDGAT1aR at their soma or axons terminals in the cNTS, elicited robust and frequency‐dependent decreases in BP and heart rate. Conversely, optical inhibition of afferents in the cNTS originating from NDGAT1aR blunted pressor responses to PE. The implication is that NDGAT1aR are sufficient and necessary to elicit appropriate compensatory responses to vascular mechanosensation induced by vasoconstriction. Opsin‐ or GCaMP6s‐expressing mice were then subjected to the DOCA‐salt model of hypertension. Optical excitation elicited hypotensive and bradycardic responses; however, the duration of these effects was altered, consistent with hypertension‐induced impairment of BR. Similarly, the increased GCaMP6s fluorescence observed after administration of PE was significantly delayed in mice that were subjected to DOCA‐salt or chronic delivery of angiotensin II. Finally, AT1aR‐flox mice were delivered AAV expressing Cre and/or tdTomato into the nodose ganglia to probe the function of AT1aR(s) themselves, in mediating the baroreflex. Preliminary results indicate deleting AT1aR(s) from the nodose ganglia alters pressor responses to vasoactive drugs. Collectively, these results reveal the structure and function of NDGAT1aR and suggest that such neurons may be exploited to discern and relieve hypertension.

Abstract 45: Sex Differences Of Renal11β-hsd1 And 11β-hsd2 Expression In A Doca-salt Model Of Hypertension.

The balance between hydroxysteroid dehydrogenase 1 (11β-HSD1) and hydroxysteroid dehydrogenase 2 (11β-HSD2) have been implicated in the regulation of mineralocorticoid effects. We have previously shown in the DOCA-salt model of hypertension that males have a more pro-inflammatory immune profile and higher BP than females. The current study tested the hypothesis that males have a higher ratio of 11β -HSD1 to 11β -HSD2 and that preventing DOCA-salt induced increases in BP significantly shifts that ratio.At 10 wks of age, male and female Sprague-Dawley rats were anesthetized and a right uni-nephrectomy (UNX) was performed. After one week of recovery, rats were randomized to the following groups: 1) UNX controls, 2) DOCA-salt (200 mg) with 0.9% saline to drink, or 3) DOCA-salt with saline + hydrochlorothiazide (HCTZ; 55 mg/kg/day) and reserpine (RES; 4.5 mg/kg/day. After 3 weeks of treatment, the remaining kidney was isolated to measure mRNA expression of 11β-HSD1 and 11β-HSD2 via RT-qPCR.Control UNX males had a higher 11β-HSD1:11β-HSD2 ratio than females (0.9 ± 0.15 vs 0.3 ± 0.04; P=0.02). After DOCA-salt, 11β-HSD1 increased in both males and females (2.5 ± 0.1 vs. 1.3 ± 0.08; P treatment <0.0001). Males maintained a higher 11β-HSD1:11β-HSD2 ratio as well has had a higher increase in 11β-HSD1 to 11β-HSD2 ratio than females (P sex =0.04; P interaction =0.04) after DOCA-salt. With HCTZ/RES treatment, 11β-HSD1 to 11β-HSD2 ratios significantly decreased in both males and females (1.9 ± 0.5 vs. 0.8 ± 0.09; P treatment =0.002), but males maintained a higher 11β-HSD1:11β-HSD2 ratio vs females (P sex =0.002; P interaction =0.54). Our data suggests that there are sex differences in the balance of renal mRNA expression of 11β-HSD1 and 11β-HSD2, with males having a greater 11β-HSD1:11β-HSD2 ratio compared to females. Preventing DOCA-salt induced increases in BP significantly shifts this ratio through an upregulation of 11β-HSD2 in both sexes.

Abstract MP04: Interleukin-33 Drives Cardiac Remodeling And Alters Macrophage Polarization In Doca-salt Hypertension

Interleukin-33 (IL-33) is an alarmin critical for alternative macrophage activation and has been linked to hypertension in humans. The goal of this study was to evaluate whether IL-33 contributes to (deoxycorticosterone acetate) DOCA-salt induced cardiac remodeling and macrophage polarization. C57Bl/6J (WT, n=8) and IL33-deficient (IL-33 KO, n=8) mice underwent uninephrectomy, implantation of a DOCA pellet, and 1% NaCl supplementation of drinking water for three weeks. A control group of WT mice (n=10) underwent a sham procedure and were maintained on normal water. Systolic blood pressure measured by tail cuff showed a significant increase in WT DOCA-salt mice (128±9 mmHg; mean±SD) and IL-33 KO DOCA-salt mice (129±11) compared to WT sham (103±11). Hypertension was accompanied by significantly increased heart to body weight ratios in WT DOCA-salt (7.0±1.0 mg/g) and IL-33 KO DOCA-salt (6.8±0.6) compared to WT sham (5.3±0.3) mice. Glomerular injury was assessed by urine albumin-to-creatinine ratios, which were similarly increased in WT DOCA-salt (0.92±0.4) and IL-33 KO DOCA-salt (0.42±0.09) mice compared to WT controls (0.03±0.003). Echocardiography was performed to assess left ventricle (LV) dimensions and systolic function. IL-33 KO DOCA-salt mice exhibited a significantly higher cardiac output (22±2 mL/min) compared to WT sham (16±3) and WT DOCA-salt (15±3) mice, driven by increased stroke volume. No significant changes in heart rate or ejection fraction were observed between groups. Additionally, IL33-KO DOCA-salt mice had an increased diastolic LV internal dimension (3.2±0.2 mm) compared to WT sham (2.7±0.3) and WT DOCA-salt (2.7±0.3). DOCA-salt induced increased mRNA expression of alternative macrophage activation markers in both LV ( Chil3, Il10) and isolated peritoneal macrophages ( Arg1, Retnla, Tgfb1 ). Peritoneal macrophages deficient in IL-33 expressed less Il10 compared to WT. Cardiac fibrosis evaluated by Masson’s trichrome showed a significant increase in LV collagen deposition in WT DOCA-salt mice (1.09±0.8% trichrome positive) compared to WT sham (0.43±0.3) but no increase in IL-33 KO DOCA-salt mice (0.54±0.3). In conclusion, IL-33 deficiency alters macrophage and cardiac function in the DOCA-salt model of hypertension.

Abstract MP57: Chronic Inhibition Of Brain Rhomboid-like Protein 2 (irhom2) Activity Decreases Arterial Blood Pressure In Salt-sensitive Hypertension In Mice

We previously reported that ADAM17 (aka tumor necrosis factor-α convertase) is critical for the development of hypertension in experimental models and patients. Recent studies highlighted that ADAM17’s formation of TNF-α relies on prior maturation of this sheddase, controlled by the rhomboid-like protein 2 (iRhom2) specifically in microglia. Genetic deletion of iRhom2 in mice shows significant attenuation of TNF-α and ADAM17 activity in a tissue specific manner. Here, we hypothesized that silencing iRhom2 activity specifically in the brain would decrease blood pressure (BP) in the DOCA-salt model of hypertension, in mice. Uninephrectomized mice were implanted subcutaneously (sc) with DOCA-pellets (50 mg) and provided with 1% saline in drinking water. In addition, mice were chronically implanted with an icv cannula connected to a sc osmotic minipump for delivery of: (1) iRhom2-siRNA (9.6 μg/kg/day), (2) scrambled siRNA (SCR 0.2 μg/kg/day), (3) ADAM17 antibody (ADAM17-Ab; 23.8 μg/kg/day) or (4) artificial cerebrospinal fluid (aCSF) for 2 weeks while BP was recorded by telemetry. DOCA-salt treatment led to a significant increase in BP in the control groups (SCR: 156 ±3 mmHg and aCSF: 161 ±1 mmHg; n=3/group; p<0.001) compared to baseline values (122 ±2 mmHg; n=12). ICV infusion of iRhom2-siRNA or ADAM17 neutralizing antibody for 2-weeks in DOCA-salt-treated mice resulted in a significant attenuation of BP (iRhom2-siRNA: 152 ±2 mmHg and ADAM17-Ab: 151 ±2 mmHg n=3/group, p<0.001). These data suggest that: 1) Selective silencing of iRhom2 from microglia is as potent as ADAM17 neutralization throughout the brain in lowering BP and 2) iRhom2 is a potential new therapeutic target for the treatment of salt-sensitive hypertension.