bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 524: Renal Hemodynamic and Excretory Responses to Infusion of Tyrosine Kinase Inhibitor, Imatinib in Anesthetized Mice
Authors: Castillo Alexander , Kadowitz Philip , Majid Dewan S
Published: July 2021

Systemic administration of a tyrosine kinase inhibitor, imatinib has been shown to have potent vasodilator activity in pulmonary and systemic vascular beds in rats. Chronic treatment with imatinib was also shown to cause marked attenuation of the renal injury in AngII induced hypertensive rats. However, the involvement of tyrosine kinases in the regulation of renal hemodynamics and excretory function is not yet clearly defined. To examine the role of constitutively active tyrosine kinases in the kidney, we have examined the renal effects of intravenous infusion of imatinib mesylate (Novartis Pharma, AG, Basel, Switzerland) at incremental doses (0.02, 0.2 and 2 mg/min/kg bw) as well as its interaction with nitric oxide (NO) activity in anesthetized mice. Systemic blood pressure (SBP) was recorded from a cannula placed in the left carotid artery. Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured by renal clearances of PAH and inulin respectively. From a control SBP value of 91 ± 3 mmHg in one group of mice (n=5), imatinib caused minimal changes in SBP at lower doses (87 ± 4 and 86 ± 5mmHg; n=5) but caused significant reduction (75 ± 4 mmHg; P<0.01) at the highest dose. During infusion of imatinib doses, there were no significant changes in RBF (from control value of 9.2 ± 0.9 to 9.2 ± 1.1, 9.9 ± 1.8 and 8.7 ± 2.2 mL/min/g kw respectively), but interestingly, there were dose-depended reductions in GFR (from a control value of 1.8 ± 0.2 to 1.7 ± 0.3, 1.5 ± 0.3, and 1.1 ± 0.3 mmHg). However, only at the highest dose, there were significant reductions in urine flow (from 6.5 ± 0.9 to 4.6 ± 0.5 μL/min/g) and sodium excretion (from 0.42 ± 0.09 to 0.17 ± 0.03 μmol/min/g) but not in fractional excretion of sodium (0.16 ± 0.04 to 0.13 ± 0.04 % ) indicating that the excretory changes were mostly dependent on hemodynamic changes. It is also observed that the usual responses (reductions in RBF, GFR etc) to the infusion of NO synthase inhibitor, nitro-L-arginine (L-NAME; 200mg/min/kg) remain intact in mice (separate group; n=5) pretreated with imatinib (0.2 mg/min/kg). These data suggest that a constitutively active tyrosine kinase pathway plays a regulatory role in maintaining glomerular filtration rate and excretory function in the kidney which seems independent of NO activity.
Abstract 543: Increased Aortic Pulse Wave Velocity in Type 2 Diabetic db/db Mice is Dependent on Blood Pressure
Authors: Anghelescu Mircea , Gooch Keith J , Trask Aaron J
Published: July 2021

Pulse wave velocity (PWV) is the gold standard for
in vivo
aortic stiffness measurements but can be dependent upon blood pressure and/or heart rate. Previous studies from our and other labs have shown increased aortic PWV in type 2 diabetic db/db mice. Moreover, preliminary
ex vivo
pressure myography data from our lab has also shown a lack of increase in passive aortic stiffness, suggesting that increased PWV
in vivo
, and therefore, increased stiffness in db/db mice may be dependent upon other mechanisms. In this study, we tested the hypothesis that increased aortic pulse wave velocity measured in db/db mice
in vivo
is blood pressure dependent under anesthesia. 16-wk old normal Db/db (n=9) and type 2 diabetic db/db (n=5) mice were anesthetized with 2% isoflurane and instrumented with two 1.2F pressure-tip catheters: one inserted in the left carotid artery and advanced to the thoracic aorta, the other inserted into the left femoral artery and advanced into the abdominal aorta. Blood pressure was continuously recorded and PWV was calculated using the foot-to-foot method. A microcannula was inserted into the right jugular vein for the administration of drugs. After a stabilization period of 25-30 mins, baseline BPs and PWVs were measured, after which mice were infused with increasing doses of phenylephrine (Phe, 100-500 nmol/kg/min) and sodium nitroprusside (SNP, 100-500 nmol/kg/min) to increase and decrease blood pressure, respectively. At baseline (prior to the infusion of any drugs), mean arterial pressure and aortic PWV were significantly elevated in db/db mice under anesthesia (MAP; Db/db: 77±5 vs. db/db: 100±4 mmHg,
p
<0.05; PWV; Db/db: 0.31±0.01 vs. db/db: 0.35±0.01 cm/ms,
p
<0.05). The increase in aortic PWV in db/db mice at baseline was completely abrogated when measured at equivalent MAPs ranging from 40-120 mmHg during the Phe and SNP infusions (
p
>0.05). In both Db/db and db/db mice, aortic PWV was significantly correlated with MAP (Db/db: r=0.94,
p
<0.001; db/db: r=0.97,
p
<0.0001). These data show that increased aortic PWV, and therefore increased aortic stiffness in db/db mice
in vivo
is dependent upon blood pressure.
Abstract 545: Modification Over Time of Pulse Wave Velocity Parallel to Changes in Both Office and 24h-ambulatory Peripheral BP as well as Aortic BP
Authors: Oliveras Anna , Segura Julián , Suárez Carmen , García-Ortiz Luis , Abad María , Gómez-Marcos Manuel , Sans Atxer Laia , Ruilope Luis M , de la Sierra Alejandro
Published: July 2021

Aim:
to assess the relationship of changes in pulse wave velocity (PWV), a marker of target organ damage, with the variation in BP over time, as assessed by three different methods of measurement: office and 24h-ambulatory peripheral BP as well as aortic BP.

Methods:
Observational prospective study in hypertensive subjects with impaired glucose metabolism consecutively recruited from Spanish Hypertension Units. Aortic BP and carotid-femoral PWV were evaluated by radial applanation tonometry (Sphygmocor®) at baseline (
b
) and after 12 months of follow-up (
fu
). Peripheral BP measurements were also recorded at baseline and at 12 months follow-up: office BP was obtained as the average of triplicate measurements taken at 1 min-intervals after 5 min of seated rest, using validated oscillometric devices; 24h-ambulatory BP recordings were taken with a validated device (Spacelabs®-90207) at 20-minute intervals throughout both the self-reported awake and asleep periods. Clinical and anthropometric features were also recorded.

PWV variation (Δ) over time was calculated as follows: Δ PWV= [(PWV
fu
- PWV
b
) / PWV
b
] x 100. BP variation over time was calculated with the same formula applied to BP values obtained with the different measurement techniques.

Correlations (Spearman “Rho”) of Δ PWV and Δ BP were calculated.

Results:
n=209 patients; mean age: 61.8 ± 11.2 y; 39% (81 of 209) were female; 80% (167 of 209) had type 2 diabetes. Other risk factors: hypertension: 100%; dyslipidemia: 69% (144 of 209); smokers: 13% (28 of 209); body mass index: 30.9 ± 4.4 Kg/m
2
. Baseline office systolic/diastolic BP (mmHg): oSBP = 143 ± 20; oDBP = 82 ± 12. Follow-up office systolic/diastolic BP (mmHg): oSBP = 136 ± 20; oDBP = 79 ± 12. Baseline PWV: 10.01 ± 3.5 m/s. Follow-up PWV: 10.19 ± 3.21 m/s.

Δ PWV correlated with: Δ oSBP (Rho=0.212; p=0.002), Δ 24h-SBP (Rho=0.254; p<0.001), Δ daytime-BP (Rho=0.232; p=0.001), Δ nighttime-BP (Rho=0.320; p<0.001) and Δ aortic-SBP (Rho=0.320; p<0.001).

Conclusion:
Modification over time of PWV, a marker of target organ damage, parallel to changes in systolic BP, both office and 24h-ambulatory peripheral BP variation as well as aortic BP variation, at 12 months of follow-up. Among them, aortic SBP and nighttime peripheral SBP both showed the best correlation.
Abstract 549: An Up-regulation Of Matrix Metalloproteinase-9 In Parietal Epithelial Cells Is Associated With A Progressive Podocyte Loss In Diabetic Glomerulopathy
Authors: Zhao Xueying , Zhang Yuanyuan , Olufade Rebecca
Published: July 2021

As a subfamily of matrix metalloproteinases (MMPs), gelatinases including MMP-2 and MMP-9 play an important role in the remodeling and homeostasis of the extracellular matrix. However, conflicting results have been reported regarding their expression level and activity in the diabetic kidney. This study investigated whether MMP-9 expression and activity were changed in glomerular epithelial cells upon albumin overload. Immunostaining, in situ zymography and Western blot for renal protein expression and MMP activity were evaluated in Zucker lean (Fa/+) and Zucker diabetic fatty (Fa/Fa) rats. Confocal microscopy revealed a focal increase in gelatinase activity and MMP-9 protein in the glomeruli of Fa/Fa rats. Increased glomerular MMP-9 staining was mainly observed in hyperplastic parietal epithelial cells expressing claudin-1 in the diabetic kidneys. Interestingly, an increase in parietal MMP-9 was often accompanied by decreased staining for podocyte markers (nephrin and podocalyxin) in the adjacent area. Additionally, urinary excretion of nephrin and podocalyxin was progressively increased in association with the levels of MMP-9 and total protein in the urine of diabetic animals. To evaluate the direct effect of albumin on expression and activity of MMP-2 and MMP-9, primary cultured rat glomerular parietal epithelial cells were incubated with exogenous albumin. MMP-9 mRNA levels were significantly increased by 306%, 565%, and 829% (n=4-6), respectively, when the cells were exposed to rat serum albumin (0.25, 0.5 and 1 mg/ml) for 24 hour. Meanwhile, albumin administration resulted in a dose-dependent increase in MMP-9 protein (7.4-, 14.1-, and 21.1-fold, respectively) and activity (3.2-, 4.5-, and 6.7-fold, respectively) in the culture supernatants of parietal epithelial cells. In contrast, albumin treatment did not significantly alter the expression level and enzymatic activity of MMP-2 in cultured parietal epithelial cells. Taken together, we have demonstrated that an up-regulation of MMP-9 in activated parietal epithelium is associated with a progressive loss of adjacent podocytes during the development of diabetic glomerulopathy. Albumin overload also induces MMP-9 production in primary cultured glomerular epithelial cells.
Abstract 556: Obese Diabetic Mice Exhibited Severe Intramuscular Lipid Accumulation after Muscle Injury
Authors: Mogi Masaki , Kohara Katsuhiko , Nakaoka Hirotomo , Kan-no Harumi , Tsukuda Kana , Wang Xiao-Li , Chisaka Toshiyuki , Kukida Masayoshi , Bai Hui-Yu , Shan Bao-Shao , Iwanami Jun , Horiuchi Masatsugu
Published: July 2021

Background:
Sarcopenic obesity, a loss of muscle and a concomitant increase in ectopic fat, is reported to increase in life-style related diseases. Especially the prevalence of sarcopenia is greater in patients with type 2 diabetes mellitus (T2DM) than in non-diabetic subjects. Increase in fat filtration enhances insulin resistance and is associated with metabolic risk factors. However, the detailed mechanism of such ectopic fat filtration in muscle of T2DM patients has not been well investigated. Here, we assessed muscle regeneration using muscle-injured models in obese diabetic mice.

Methods:
Male eight-week-old wild-type mice (C57BL6) and T2DM mice (KKAy) were undergone intramuscular injection of cardiotoxin (Ctx) (100uL/10uM) into tibia. After two weeks, muscle were removed and stained with hematoxylin and eosin. KKAy replaced by bone marrow prepared from GFP-transgenic mice were generated by 8 Gy whole body X-ray irradiation. Cell-differentiation was evaluated by immunofluorescent analysis. Some mice were treated with all trans-retinoic acid (ATRA) and PPAR-gamma antagonist, GW9662.

Results:
In 26 week-old KKAy, magnetic resonance imaging analysis showed that intramuscular fat deposition was estimated as higher intensity compared with C57BL6 mice. Treatment with Ctx showed a significant increase in honey comb structure which was perilipin positive. KKAy mice exhibited impaired muscle regeneration. Tibial muscle weight was approximately a half in Ctx-injured KKAy. Such change was also observed in another diabetic mouse model, db/db, but not in streptozocin-induced diabetic mice. Fat formation was remarkably increased in aged KKAy compared with younger mice. Several fat formation was GFP-positive in GFP-chimeric mice indicating that fat tissue was partially generated from marrow cells. Fat formation was desmin-negative and vimentin-positive indicating that these are mesenchymal origin. Treatment with ATRA reduced fat filtration. On the other hand, treatment with GW9662 enhanced fat filtration.

Conclusion:
Obese diabetic mice showed significantly impaired muscle regeneration with replacement of fat deposition, suggesting that diabetes enhanced sarcopenic obesity possibly due to inhibitory effect of cell differentiation.
Abstract 609: Differential Effects of Rapamycin Treatment on Cardiac Pathology in Lean and Obese Rats
Authors: Luck Christian , DeMarco Vincent , Slusarz Anna , Lum-Naihe Kelly , Pulakat Lakshmi
Published: July 2021

Rapamycin (Rap) is a widely-used drug in organ transplant prophylaxis, drug eluting stents to prevent coronary restenosis, and cancer therapy. Rap is also considered as an anti-aging drug that improves health and life-span. However, evidence also suggests that new-onset diabetes mellitus (DM) is an adverse outcome of Rap treatment. Since DM is an independent risk factor for heart disease, it is unclear whether chronic Rap treatment would be beneficial or detrimental for myocardium. To test the hypothesis that chronic Rap treatment would have differential effects on cardiac pathology in lean and obese insulin resistant individuals, we investigated the effect of 12-week Rap treatment (via subcutaneous implantation of Rap pellets delivering Rap at a concentration of 750μg/kg/day) on young (8-20 weeks), Zucker lean (ZL) rats and insulin resistant Zucker obese (ZO) rats. While Rap treatment significantly suppressed body weight, heart weight, fasting plasma levels of cholesterol, triglycerides, and LDL in ZO rats (p<0.05), it also worsened DM as indicated by severe hyperglycemia, polydipsia and polyuria (p<0.05). Conversely, Rap-treated ZL rats did not develop DM. Echocardiography showed that 14-week old ZO rats exhibited abnormalities in diastolic parameters, including decreases in the tissue doppler E’/A’ ratio, mitral inflow propagation velocity (Vp) indicative of impaired active phase relaxation, as well as increases in LV filling pressure (E/E’ and E/Vp ratios), isovolumic relaxation time (IVRT) and myocardial performance index (Tei index of global cardiac function) compared to age-matched ZL rats. With the exception of IVRT, these abnormalities were improved in ZO rats treated for 6 weeks with Rap. However, an additional 6 weeks of Rap treatment reversed this improvement in diastolic abnormalities. IVRT, Vp and E/Vp ratio tended to be more impaired in ZO rats with 12 week Rap treatment, although none of the trends reached statistical significance. Histopathology analysis revealed that Rap treatment induced significant cardiomyofibril disarray in both ZL and ZO rats (p<0.05). These observations suggest that Rap treatment advances DM in obese, insulin resistant rats and induces cardiac structural damage in both healthy and obese rats.
Abstract 649: Endothelial-derived Sphingolipids Preserve Systemic Vascular Function And Blood Pressure
Authors: Cantalupo Anna , Zhang Yi , Obinata Hideru , Galvain Syvain , Jiang Xiang-Cheng , Giordano Frank , Hla Timothy , Di Lorenzo Annarita
Published: July 2021

Endothelial dysfunction is a critical event in many cardiovascular diseases including hypertension. Although lipid signaling is implicated in endothelial dysfunction and cardiovascular diseases, specific molecular mechanisms are poorly understood. Here we report a novel regulation of endothelial sphingolipid synthesis by Nogo-B, membrane protein of the endoplasmic reticulum that modulates local sphingolipid production with direct effects on vascular function and blood pressure. Nogo-B inhibits serine palmitoyltransferase, rate-limiting enzyme of the sphingolipid de novo synthesis, controlling endothelial sphingosine 1-phosphate production and its autocrine G-protein-coupled receptor-dependent signaling actions. Mice lacking Nogo-B are hypotensive (90.1±1.6 vs. 119.9±2.6 mmHg WT mice), resistant to Ang-II (500ng/Kg/min)-induced hypertension (150.4±2.5 vs. 108.4±1.5 mmHg, compared to WT mice, 24 days after AngII infusion), and preserve endothelial function and nitric oxide release. Pharmacological inhibition of serine palmitoyltransferase with myriocin in mice that lack Nogo-B reinstated endothelial dysfunction and Ang-II-induced hypertension (143.9±1.5 vs. 90.1±1.6 mmHg, myriocin vs. vehicle treated Nogo-A/B-/- mice).

Our study identifies Nogo-B as a key inhibitor of local sphingolipid synthesis and indicates that autocrine sphingolipids signaling within the endothelium are critical for vascular function and blood pressure homeostasis.
Abstract MP05: Mineralocorticoid And Angiotensin II Type 1 Receptors In The Subfornical Organ Mediate Angiotensin II Hypertension In Rats
Authors: Wang Hong-Wei , Huang Bing S , Chen Aidong , Ahmad Monir , Leenen Frans H
Published: July 2021

Circulating Ang II increases BP through both peripheral and central mechanisms. Activation of both AT
1
R and MR in the CNS plays a critical role in Ang II-induced hypertension. The subfornical organ (SFO) contains both MR and AT
1
R and can relay the signals of circulating Ang II to downstream nuclei such as the PVN. In this study, we evaluated the effect of intra-SFO infusion of AAV-MR-siRNA or AAV-AT
1a
R-siRNA on MR or AT
1
R mRNA expression in the SFO and PVN and Ang II induced hypertension as assessed by telemetry. Two week sc infusion of Ang II at 500 ng/kg/min in Wistar rats on regular salt diet increased BP by 60~65 mmHg. This pressor effect of Ang II was largely prevented by AAV-MR-siRNA or AAV-AT
1
R-siRNA in the SFO (Figure). Ang II increased AT
1
R mRNA expression (1.7± 0.4 vs 0.8 ± 0.04 х10
-1
, p<0.05) in the SFO. Both MR- and AT
1
R-siRNA in the SFO prevented this increase (1.1 ± 0.2х10
-1
and 1.0± 0.3х10
-1
). In contrast, Ang II decreased MR mRNA expression (3.2 ± 0.2 vs. 4.1 ± 0.2х10
-2
, p<0.05) in the SFO. Both MR- and AT
1
R-siRNA further decreased MR mRNA expression (2.7 ± 0.2 х10
-2
and 2.3 ± 0.3 х10
-2
). In the PVN, Ang II increased AT
1
R mRNA by ~100% and MR mRNA by ~20%. Only AT
1
R-siRNA in the SFO prevented these increases in the PVN. AAV mediated eGFP fluorescence expression was observed in the SFO but not the PVN. These results suggest that circulating Ang II differentially regulates MR and AT
1
R expression in the SFO. Prevention of Ang II induced AT
1
R up-regulation by knockdown of either AT
1
R or MR suggests a MR dependent regulation of AT
1
R in the SFO. MR-AT
1
R signaling in the SFO appears to play a major role in Ang II-induced hypertension in rats.

* p<0.05, vs. baseline values; a: p<0.05, vs. AAV-SCM-siRNA
Abstract MP10: Early Functional and Structural Alterations of Resistance Arteries in Mice Treated with an Inhibitor of the Vascular Endothelial Growth Factor Receptor
Authors: SAVOIA CARMINE , Arrabito Emanuele , Montezano Augusto C , Nicoletti Carmine , Small Heather Y , Sada Lidia , Rios Francisco , Volpe Massimo , Touyz Rhian M
Published: July 2021

Background:
Inhibition of tyrosine kinases receptors such as vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) improves outcomes in patients with cancers. Only VEGFR inhibitors, however, induce severe hypertension whose mechanisms remain unclear. We hypothesized that VEGFR inhibitors may induce early vascular functional and structural alterations, that may precede the development of hypertension.

Methods and results:
Normotensive SV-129 mice (8 weeks old, 5 for each group) were treated or not with the VEGFR inhibitor Vatalanib (VAT, 100 mg/Kg/day) or the EGFR inhibitor Gefitinib (GEF, 100 mg/Kg/day). Vehicle-treated control mice were also studied. Blood pressure (BP) was measured by tail-cuff method. Endothelium-dependent and -independent relaxations were assessed by concentration-response curves to acetylcholine (1 nM to 100 μM) ± L-NAME (100 μM) and sodium nitroprusside (10 nM to 1 mM) respectively, in mesenteric arteries pre-contracted with norepinephrine (10 μM). Media-to-lumen ratio (M/L, an index of early vascular remodeling), and cross sectional area (CSA) were evaluated on pressurized preparations.

After two weeks, BP was similarly preserved in both VAT- and GEF-treated mice as compared to vehicle-treated mice (89.8±1.5 mmHg and 87.2±2.8 mmHg vs 92.2±2.2 mmHg, respectively, NS). Endothelium-dependent relaxation was similarly preserved in vehicle-treated and GEF-treated mice, whereas it was reduced in VAT-treated mice (-17% vs vehicle-treated mice, P<0.05). L-NAME blunted acetylcholine-induced relaxation in all groups except in VAT-treated mice, suggesting an impairment of NO production only in this group. Endothelium-independent relaxation was similar in all groups. Only VAT-treated mice presented increased M/L as compared to vehicle-treated mice (6.3±0.1% vs 5.4±0.1%, P<0.05). M/L resulted similar in GEF-treated and vehicle-treated mice. CSA was similar in all groups.

Conclusion:
In normotensive mice, only VAT promoted early vascular alterations such as endothelial dysfunction and vascular remodeling in resistance arteries. Those changes in the vasculature are distinctive of hypertension and might precede and sustain the development of the hypertensive disease.
Abstract 016: Profound and Sustained Amplification of Circulating ACE2 Activity Does Not Protect Diabetic Mice from Kidney Disease
Authors: Wysocki Jan , Ye Minghao , Khattab Ahmed M , Kanwar Yashpal , Osborn Mark , Batlle Daniel
Published: July 2021

ACE2 is a monocarboxypeptidase that by converting AngII to Ang1-7 should down-regulate the renin-angiotensin system and therefore provide a means to therapeutically target diabetic kidney disease, a condition where the kidney RAS is overactive. Previous work indicated that soluble human recombinant (r)ACE2 administration for 4 weeks attenuated kidney injury in diabetic Akita mice. Whether such effect of rACE2 can be confirmed and attributed to augmented ACE2 activity is uncertain because chronic use of human rACE2 in mice induces immunogenicity and the development of antibodies that neutralize serum ACE2 activity.

To examine the effect of chronic amplification of circulating ACE2 on kidney injury caused by STZ-induced diabetes and to circumvent the immunogenicity arising from xenogeneic ACE2, ACE2 of mouse origin was administered to mice using either daily i.p. injections (1 mg/kg) of mrACE2 for 4 weeks or after 20 weeks of ACE2 mini-circle (MC) (10-30ug/mouse) administration. MC provides a form of gene delivery that is resistant to gene silencing and, in addition, greatly optimizes long-term in vivo overexpression of proteins of interest. ACE2MC resulted in a profound and sustained increase in serum ACE2 activity (2.4±0.3 vs. 497±135 RFU/ul/hr, p<0.01) but kidney ACE2 activity was unchanged (17.4±1.3 vs. 19.0±0.8 RFU/ug prot/hr). mACE2-treated mice injected with STZ developed diabetes similar to sham mice injected with STZ. Systolic BP was not different between non-diabetic mice, sham STZ-mice, and STZ-mice receiving mACE2 by either i.p. mrACE2 or ACE2MC. Urinary albumin was similarly increased in sham STZ-mice and in STZ-mice receiving mACE2. Glomerular mesangial score and glomerular cellularity were both increased to a similar extent in sham STZ-mice and in STZ-mice with mACE2 administration, as compared to non-diabetic controls.

In conclusion, profound and long-term augmentation of ACE2 activity confined to the circulation is not sufficient to attenuate glomerular pathology and albuminuria in STZ-induced diabetic kidney disease probably because of lack of kidney delivery of ACE2. Strategies to achieve over-expression of ACE2 at the kidney level are needed to demonstrate a beneficial effect of this enzyme on diabetic kidney disease.
Abstract 028: ACE2 Activator, Diminazene, Rebalances Gut Microbial Dysbiosis and Attenuates Pulmonary Hypertension
Authors: Shenoy Vinayak , Yang Tao , Rubiano Andres , Guzzo Dominic , Horowitz Alana , Santisteban Monica , Rathinasabapathy Anandharajan , Simmons Chelsey , Katovich Michael , Raizada Mohan
Published: July 2021

Introduction:
Our previous studies have established that increasing the levels of pulmonary Angiotensin converting enzyme2 (ACE2) either by genetic overexpression or by a small molecule activator, Diminazene aceturate (DIZE) provides protection against lung injury. In view of the mounting evidence of the involvement of the gut microbiota in inflammatory, metabolic and neurological diseases, we proposed the following hypothesis: gut dysfunction and microbial dysbiosis is associated with pulmonary hypertension (PH) and that the cardiopulmonary beneficial effect of DIZE is mediated, in part, by its influence on the gut microbial composition.

Methods:
PH was induced in male Sprague Dawley rats by a single injection of monocrotaline (MCT; 50mg/Kg s.c). A subset of MCT rats was treated daily with DIZE (15mg/Kg/day s.c) for 4-weeks, after which hemodynamic parameters were measured and fecal samples collected for bacterial 16S ribosomal DNA analysis. In addition, colon samples were isolated to determine tissue stiffness by ex vivo atomic force microscopy.

Results:
MCT administration resulted in the development of PH as evidenced by increase in right ventricular systolic pressure (RVSP - Control: 30+2; MCT: 93+10 mmHg; p<0.05), which was associated with significant decreases in gut microbial richness (37%), diversity (22%), and evenness (16%). Furthermore, we observed a significant reduction in acetate- and butyrate-producing, and increases in lactate-producing bacterial population in PH animals. Elastic modulus and viscosity of the colon were increased by 61% and 86% respectively in MCT animals as compared with controls, indicating greater tissue stiffness (Elastic modulus - Control: 7.33+1.66; MCT: 11.84+3.41 kPa; Viscosity - Control: 145+19; MCT: 270+36 kPa*s; p<0.05). However, chronic treatment with DIZE attenuated all these parameters (RVSP - MCT+DIZE: 52+9 mmHg; Elastic modulus - MCT+DIZE: 8.97+3.84 kPa; Viscosity - MCT+DIZE: 180+77 kPa*s; p<0.05)

Conclusions:
These observations demonstrate for the first time that a) gut microbial dysbiosis is associated with MCT-induced PH; b) DIZE attenuates PH pathophysiology and significantly rebalances dysbiosis. They suggest that the gut microbiota could be a potential target for PH therapy.
Abstract 044: Pappa2 is Linked to Salt-sensitive Hypertension in Dahl SS Rats
Authors: Cowley Allen W , Yang Chun , Kumar Vikash , Lazar Jozef , Jacob Howard , Geurts Aron M , Liu Pengyuan , Dayton Alex , Kurth Theresa , Liang Mingyu
Published: July 2021

The goal of the present study was to narrow a 1.37 Mbp region containing five genes on chromosome 13 (positions 80.92-82.29 Mbp in the Rn5 genome assembly) that we have previously found to influence the mean arterial pressure (MAP) by at least 25 mmHg in SS rats fed a high salt diet (8.0% NaCl for 14 days). The creation of 6 overlapping subcongenic strains that cover this region identified a 0.71 Mbp region (positions 81.01-81.72 Mbp) in which substitution of SS alleles with BN alleles reduced salt-induced hypertension in congenic SS rats by nearly 30 mmHg and significantly reduced urinary albumin excretion (UalbV) (see figure below; n=10-15/strain; * significant difference from SS; p<0.05). Analysis of the narrow candidate region revealed the presence of two protein-coding genes (
Pappa2
and
Astn1
) and a microRNA (
miR488
), none of which are known to be mechanistically involved in hypertension.
Pappa2
mRNA in these rat strains fed 0.4% NaCl diet was expressed at 6-10 fold higher levels in the renal cortex of the salt-resistant congenic strains with the BN allele (26-N, -O, and [[Unable to Display Character: –]]P) compared to strains with the SS allele (26-Q, -R, and [[Unable to Display Character: –]]S). A
Pappa2
coding sequence variant of unknown functional importance was identified. Immunohistochemistry and fluorescence
in situ
hybridization studies localized
Pappa2
to intercalated cells of the cortical and medullary collecting duct.
Astn1
and
miR-488
were not differentially expressed in renal tissue. Together, these findings point towards
Pappa2
as a candidate gene for salt-induced hypertension in SS rats.
Abstract 065: Expression of a Dominant Negative Cullin 3 Mutant (Cul3[[Unable to Display Character: ∆]]9) Impairs Endogenous Cullin 3 Activity and Causes Impaired Vasorelaxation of Aorta
Authors: Agbor Larry N , Ibeawuchi Stella-Rita C , Quelle Frederick W , Sigmund Curt D
Published: July 2021

Patients with dominant loss-of-function mutations in Cullin 3 (Cul3) resulting in deletion of exon 9 (Cul3[[Unable to Display Character: ∆]]9) exhibit severe early onset hypertension correlated with reduced degradation of Cul3 substrates. We have shown that knockout of Cul3 induced by clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 in HEK293T cells resulted in accumulation of the Cul3 substrate RhoA. To gain insight into the mechanism of attenuated Cul3[[Unable to Display Character: ∆]]9-mediated protein degradation, we utilized CRISPR-Cas9 genome editing to generate Cul3[[Unable to Display Character: ∆]]9-expressing cell lines. We hypothesized that expression of the Cul3[[Unable to Display Character: ∆]]9 protein will impair endogenous Cul3 wildtype (WT) function and accumulate Cul3 substrates. CRISPR guide RNAs targeting endogenous Cul3 were cloned into a plasmid expressing humanized Cas9. HEK293T cells were transfected and single cell clones selected. Sequencing revealed that genome editing induced a 548 bp deletion spanning exon 9 (Chromosome 2:224503433-224503979). Sequencing of the mRNA transcript also revealed that exon 9 was deleted, and Cul3[[Unable to Display Character: ∆]]9 mRNA and protein was expressed. Although RhoA protein levels were normal, Cul3 substrates WNK4 and PLK1 were significantly increased in Cul3[[Unable to Display Character: ∆]]9-expressing cells (WNK4: 1.5±0.12 Cul3[[Unable to Display Character: ∆]]9 vs 1.0±0.02 WT; PLK1: 1.4±0.06 Cul3[[Unable to Display Character: ∆]]9 vs 1.0±0.03 WT, P<0.001). Interestingly, Cul3[[Unable to Display Character: ∆]]9-expressing cells exhibited impaired Cul3 ubiquitin ligase activity compared to WT. We also generated a novel transgenic mouse model inducibly expressing Cul3[[Unable to Display Character: ∆]]9 protein specifically in smooth muscle (termed S-Cul3[[Unable to Display Character: ∆]]9) and assessed vascular responses in the aorta using a wire myograph. Aorta from S-Cul3[[Unable to Display Character: ∆]]9 transgenic mice exhibited impaired ACh relaxation compared to WT (at 30 μM: 55±2% sCul3[[Unable to Display Character: ∆]]9 vs 71±7% WT, p<0.0001). In contrast, vasodilation to the nitric oxide donor, nitroprusside (SNP) was normal, as was constriction to endothelin-1. There was a significant increase in RhoA protein expression in aorta of S-Cul3[[Unable to Display Character: ∆]]9 transgenic mice compared to WT (1.6±0.2 sCul3[[Unable to Display Character: ∆]]9 vs 1.0±0.1 WT, P<0.05). These findings suggest a mechanism whereby Cul3[[Unable to Display Character: ∆]]9 protein may interfere with endogenous Cul3 and impair the degradation of Cul3 substrates such as RhoA or WNK4, contributing, at least in part-, to hypertension.
Abstract 075: Proinflammatory T Helper 17 Cells as an Indicator of Hypertension
Authors: Kim Seungbum , Rodriguez Vermali , Pepine Carl J , Raizada Mohan K
Published: July 2021

Objectives:
T cells and interleukin 17 (IL17) have been shown to be critical in the development of hypertension (HTN). This implicates a significant role of IL17 producing T helper 17 (Th17) cells in HTN. Thus, the objective of our study was to investigate if Th17 cell levels in the peripheral blood (PB) and the bone marrow (BM) are correlated with blood pressure.

Methods:
Saline or Ang II was chronically infused into C57BL6 mice (1000ng Ang II/kg/min using osmotic pumps) for 3 weeks. This resulted in an increase in MAP of 45±10 mmHg. BM and PB from saline and Ang II-treated mice were analyzed using FACS. A parallel human study was conducted using blood samples obtained from hypertensive patients (n = 8, systolic BP ≥125 mmHg) and normotensive subjects (n = 10, systolic BP <125 mmHg). FACS analysis was used to examine changes in the inflammatory cells levels in these patients.

Results:
We observed 87% and 36% increases in both Sca-1
+
c-Kit
+
Lin
-
hematopoietic stem cell (HSC; 0.23±0.04% vs. 0.43±0.05%) and Sca-1
+
c-Kit
-
Lin
-
lymphoid progenitors (10.8±1.1% vs. 14.7±2.9%) in the BM of Ang II infused mice. These are upstream stem/progenitor cells for T cells. This was associated with 30% and 190% increases in CD4
+
IL17
+
Th17 cells in the BM and PB (1.20±0.09% vs. 1.61±0.19 % and 4.8±2.0 % vs. 14.7±3.8 % respectively) in the Ang II infused mice. Importantly, there were 58% and 206% increases of angiotensin II type 1 receptor (AT1R) expressing CD4
+
T cells in the BM and PB of Ang II HTN mice (0.40±0.04 % vs. 0.63±0.14 % and 1.1±0.2 % vs 3.4±1.1 % respectively) and ~ 85% of these cells were also positive for IL-17. Consistent with mouse data, analysis of PB showed a 470% increase of Th17 cells in HTN patients (0.48±0.18 % vs 2.72±1.2%).

Conclusions:
We observed (i) Increased hematopoietic stem/progenitor cells in Ang II HTN mice; (ii) increased Th17 cells in both HTN mice and humans and (iii) the majority of AT1R expressing CD4
+
T cells was Th17 cells. Taken together, these observations indicate that Th17 cells may be an important indicator of those destined to develop HTN and suggest that these cells may represent a novel therapeutic target.
Abstract 095: Human (Pro)renin Receptor Activation Induces an Angiotensin II-Independent Pressor Response Mediated by NADPH Oxidase 4 Activity
Authors: Sullivan Michelle N , Li Wencheng , Sigmund Curt D , Feng Yumei
Published: July 2021

The binding of prorenin to the (pro)renin receptor (PRR) induces non-proteolytic activation of prorenin and generation of angiotensin II (Ang II). PRR activation can also induce Ang II-independent signaling pathways. However, whether Ang II-independent signaling pathways are critical for blood pressure (BP) regulation is not known. To address this question, we created transgenic mice that overexpress the human PRR (hPRR) selectively in neurons (Syn-hPRR). Activated human prorenin (hPRO) cannot cleave endogenous mouse angiotensinogen to generate Ang II. Therefore, administration of hPRO to Syn-hPRR mice can be used to examine Ang II-independent PRR signaling in BP regulation. Intracerebroventricular (ICV) infusion of hPRO increases BP in Syn-hPRR mice (ΔMAP 23 ± 4.6, n = 4) but has no effect on wildtype (WT) mice (ΔMAP 2 ± 0.8, n = 6). The hPRO-induced pressor response in Syn-hPRR mice is unaffected by co-infusion with the Ang II type 1 receptor blocker losartan (ΔMAP 19 ± 5.2, n = 8), suggesting that the response is independent of Ang II. Interestingly, co-infusion with an inhibitor of the reactive oxygen species-generating enzyme NADPH oxidase (NOX), diphenyleneiodonium, nearly abolishes the hPRO-induced pressor response in Syn-hPRR mice (ΔMAP 4.7 ± 1.0, n = 4), indicating that NOX activity is required. Additionally, we find that basal NOX activity is enhanced in the Syn-hPRR hypothalamus relative to WT mice (1.4 fold change). We next examined which NOX isoform is responsible for the hPRO-induced pressor response and enhanced activity. NOX4 mRNA levels are greater (2.7 ± 0.6 fold change), but NOX1 (1.2 ± 0.3 fold change) and NOX2 (1.2 ± 0.3 fold change) mRNA levels are not different, in the hypothalamus of Syn-hPRR compared to WT mice (n = 3). Adenovirus-mediated delivery of NOX2, NOX4, or a scrambled sequence shRNA was ICV injected in Syn-hPRR mice. After 7 days, we found that treatment with NOX2 (ΔMAP 20 ± 5.2) or scrambled (ΔMAP 23 ± 3.2) shRNA had no effect on the hPRO-induced pressor response (n = 5). However, the hPRO-induced increase in BP is attenuated in Syn-hPRR mice injected with NOX4 shRNA (ΔMAP 5.9 ± 2.8). Together, these data indicate that NOX4 mediates the Ang II-independent pressor response to activation of the human (pro)renin receptor in Syn-hPRR mice.
Abstract 097: Vascular Aging in Aldosterone Associated Hypertension: Role of NADPH Oxidase 1
Authors: Harvey Adam , Montezano Augusto C , Hood Katie Y , Lopes Rheure A , Graham Delyth , Touyz Rhian M
Published: July 2021

The vascular phenotype in hypertension is characterised by features typically observed in the ageing vasculature. Pathophysiological processes underlying premature vascular aging in hypertension remains unclear but aldosterone (aldo) and oxidative stress may be important. We postulated that physiological aging is amplified in hypertension due to increased aldo-induced Nox activation and redox signalling. We used arteries from adult WKY (18 weeks), aged WKY (52 weeks) and adult stroke-prone spontaneously hypertensive (SHRSP) rats. Blood pressure was measured by tail-cuff. Vascular function/structure was analysed by myography. Gene level was assessed by qPCR and protein by immunoblotting. BP was increased in SHRSP (180.7±2.5 vs. 127±2.7 mmHg, p<0.05). Endothelial dysfunction was observed in vessels from SHRSP. Increased vascular contraction in aged WKY rats was similar to SHRSP rats (p<0.05 vs WKY). Increased vascular stiffness was observed in arteries from aged WKY and SHRSP compared to WKY rats. Nox2 (0.82±0.4/2.4±0.9 vs 0.22±0.2), NoxA1 (4.9±2/9.5±5 vs 1±0.3) and NoxO1 (1.9±0.6/4.1±1 vs 1±0.4) mRNA was increased (p<0.05; SHRSP/aged WKY vs WKY). Nox1 mRNA (2.3±0.8 vs 1.1±0.4) was only increased in SHRSP rats (p<0.05; vs WKY). Similarly, mRNA levels of MCP-1 (2.3±0.5/3.9±1.9 vs 0.3±0.1) and RANTES (7.4±2/6.3±1.7 vs 1.1±0.2), aging-related inflammatory markers, and cell cycle inhibitors, p21 (3.2±1.1/3.1±0.7 vs 1±0.1) and p27 (2.2±0.7/2±0.8 vs 0.4±0.1), were increased in SHRSP and aged WKY rats (p<0.05; SHRSP/aged WKY vs WKY). ROS production (VSMC: 1.74±0.4 AU/protein), H2AX (DNA damage; 1.3±0.1) and aldosterone (plasma; 99.5±19 pg/mL) levels were increased in SHRSP rats (p<0.05; vs WKY). Aldo-induced Nox1 mRNA expression and p66SHC activation was exacerbated in VSMCs from SHRSP rats; an effect blocked by ML171 (a Nox1 inhibitor) and blunted in VSMCs from Nox1 KO mice. In conclusion, endothelial dysfunction and vascular remodelling in hypertension are associated with increased aldo-mediated activation of pro-inflammatory and redox-sensitive pathways. These processes involve Nox1. Our findings identify an important role for aldo/Nox1/ROS in molecular processes underlying vascular changes of ageing in hypertension.
Abstract 103: Increased Blood Pressure Drives Renal T-cell Infiltration in the Dahl Salt-Sensitive Rat
Authors: Evans Louise C , Petrova Galina , Kurth Theresa , Mattson David L , Cowley Allen W
Published: July 2021

Previous studies have shown that renal T-cell infiltration is a key component of salt-sensitive hypertension in Dahl salt-sensitive (SS) rats. Here we used chronic servo control experiments to determine the contribution of renal perfusion pressure (RPP) to T-cell infiltration in the SS rat kidney. An aortic balloon occluder was placed around the aorta, between the renal arteries, and used to maintain blood pressure to the left kidney at control levels, ~128 mmHg, during 7-days of salt induced hypertension. During the same period, the right kidney was exposed to increased RPP, averaging 158 ± 4 mmHg by high salt (4% NaCl) day-7. The number of infiltrating T-cells was compared between the two kidneys. Renal T-cell infiltration was significantly blunted in the left-servo controlled kidney compared to the right-uncontrolled kidney. The number of mature (CD3+), helper (CD3+CD4+) and cytotoxic T-cells (CD3+CD8+) were all significantly lower in the servo controlled kidney than in the hypertensive kidney (Fig.1). This effect was not specific to T-cells, monocyte, macrophage and B-cell infiltration were all significantly exacerbated in the hypertensive kidney. Increased RPP was also associated with augmented renal injury, with increased protein casts and glomeruli damage in the hypertensive kidney. We conclude that during the development of salt-sensitive hypertension increased RPP contributes to renal T-cell infiltration in SS rats; when blood pressure is maintained at control levels T-cell infiltration is significantly attenuated in the servo-controlled kidney relative to the hypertensive kidney despite exposure to comparable sympathetic drive and circulating factors.
Abstract 138: High Salt Intake Augments Excitability of Pre-sympathetic PVN Neurons Through Dysfunction of the Endoplasmic Reticulum Ca2+ ATPase
Authors: Larson Robert A , Chapp Andrew D , Huber Michael J , Cheng Zixi , Shan Zhiying , Chen Qing-Hui
Published: July 2021

High salt (HS) intake sensitizes pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) leading to augmented neuronal excitability. Recently, we reported that dysfunction of Ca
2+
dependent K
+
channels in the PVN contributes to HS intake induced sympathoexcitation. The endoplasmic reticulum (ER) acts as a Ca
2+
store and plays an important role in regulating intracellular Ca
2+
homeostasis. The ER Ca
2+
ATPase is responsible for maintaining the high level of ER Ca
2+
and loss of function would deplete the Ca
2+
store contributing to the reduced activity of Ca
2+
dependent K
+
channels. We hypothesized that a 2% (NaCl) HS diet for 5 weeks would reduce function of the ER Ca
2+
ATPase and augment excitability of PVN neurons with axon projections to the rostral ventrolateral medulla (PVN-RVLM) identified by retrograde label. In whole cell current-clamp recordings from PVN-RVLM neurons, graded current injections evoked graded increases in spike frequency. Maximum discharge was evoked by +200 pA injections and averaged 22±2 Hz (n=6) in normal salt (NS) control and was significantly augmented (p<0.05) by HS diet 34±5 Hz (n=8). Bath application of thapsigargin (TG) (0.5 μM), the ER Ca
2+
ATPase inhibitor, augmented excitability of PVN-RVLM neurons in NS (32±4 Hz, n=5, p<0.05), yet had no significant effect in HS rats (32±6 Hz, n=6). ER Ca
2+
ATPase function was assessed in whole animal preparations by bilateral PVN microinjection of TG in anesthetized rats. PVN microinjection of TG (0.15, 0.3 0.75 and 1.5 nmol/100nl) increased sympathetic nerve activity (SNA) and mean arterial pressure (MAP) in a dose-dependent manner in NS rats. Maximum increases in splanchnic SNA (SSNA), renal SNA (RSNA) and MAP elicited by PVN TG (0.75 nmol/100nl; n=5) were 93±7%, 75±7%, and 11±2mmHg, respectively. In contrast, sympathoexcitatory responses to PVN TG (0.75 nmol/100nl; n=5) were attenuated in HS treated rats (SSNA 41±8%, RSNA 22±5%, p<0.05 vs. NS) while MAP responses demonstrated no significant difference (+8±2 mmHg, p>0.05 vs NS). Our data indicate that a HS diet reduces ER Ca
2+
ATPase activity and augments excitability of PVN-RVLM neurons in vitro. Altered ER Ca
2+
homeostasis may contribute to sympathoexcitation through loss of Ca
2+
dependent K
+
channel activity in the PVN.
Abstract MP12: The AA2-Ratio: A Novel Screening Test for Primary Aldosteronism in Hypertensive Patients
Authors: Poglitsch Marko , Schwager Cornelia , van Oyen Dunja , Aigner Claudia , Domenig Oliver
Published: July 2021

Primary aldosteronism (PA) is severe form of hypertension characterized by a strongly increased aldosterone secretion mediated by adenomas or other forms of adrenal hyper-activity. Once detected, PA can be usually cured by either surgical intervention or by appropriate pharmacologic treatments. This is also reflected in clinical guidelines of Endocrine Societies in Europe and the US, suggesting extensive PA screening activities among resistant hypertensive patients. The incidence of PA among hypertensive patients varies strongly between different studies, which is in part caused by the complex state-of-the-art testing procedure that unfortunately is far away from being a versatile PA screening tool. Despite strong limitations regarding selectivity, sensitivity and the interference with multiple anti-hypertensive drugs, the aldosterone-renin-ratio (ARR) is widely used for PA case detection. However, there is still a strong demand for accurate and reliable and patient friendly PA case detection. The use of novel and more accurate technologies for quantification of aldosterone and renin activity might help to improve the power of the ARR as a diagnostic tool for PA. However, there is a big need for a versatile PA screening assay that doesn’t interfere with anti-hypertensive treatments and therefore allows the clear identification of PA patients without complex corrections and adaptions being necessary and without increasing the patient’s cardiovascular risk in the course of the diagnostic process. The Aldosterone-to-Angiotensin-II-Ratio (AA2-Ratio) is a novel mass-spectrometry based high-throughput test for PA that combines the plasma levels of aldosterone and physiologically active angiotensin II into a diagnostic ratio. The test performance is superior to the ARR in terms of the diagnostic window and method accuracy. The AA2-Ratio does not interfere with standard anti-hypertensive drugs including ACE inhibitors. First data obtained in a proof-of-concept study investigating PA positive and negative patients proved the AA2-Ratio to be a powerful and cost-effective diagnostic tool for the diagnosis of PA in clinical practice.
Abstract MP13: Etamicastat Prevents Arterial Blood Pressure Increase in Mice Lacking Salt-inducible Kinase 1 (SIK1)
Authors: Pires Nuno , Igreja Bruno , Moura Eduardo , Bonifácio Maria João , Serrão Paula , Soares-da-Silva Patrício
Published: July 2021

Loss of salt-inducible kinase 1 (SIK1) triggers an increase in blood pressure (BP) upon a chronic high-salt intake in mice (Circ Res 2015;116:642-52). Here, we address possible acute mechanisms that may relate to the observed high BP in mice lacking SIK1. SIK1 knockout (

sik1
-/-

) and wild-type (

sik1
+/+

) littermate mice were challenged for seven days with a normal- (0.3% NaCl) or high-salt (8% NaCl) diet. Systolic BP (SBP) was significantly increased in
sik1

-/-

mice (137.0±17.2 mmHg) after seven days of high-salt intake, as compared to

sik1
+/+

mice counterparts (120.6±4.5 mmHg). The renin-angiotensin system (RAS) and the sympathetic nervous system (SNS) were assayed in order to investigate the possible causes for the increase in SBP in

sik1
-/-

mice fed a high-salt diet. No differences in renin (normal-salt: 463.4±17.9, high-salt: 462.9±28.9 pg/ml) and angiotensin II (normal-salt: 45.8±10.0, high-salt: 39.0±8.5 pg/ml) serum levels were observed. The activity of dopamine β-hydroxylase (DβH), the enzyme that converts dopamine (DA) to norepinephrine (NE), was significantly increased in the adrenal glands of

sik1
-/-

mice fed a high-salt diet (356.7±32.8 nmol/mg protein) as compared to

sik1
-/-

mice on a normal-salt diet (184.4±14.4 nmol/mg protein). Similarly, urinary catecholamines (DA, NE, epinephrine) and L-DOPA were significantly increased (3- to 7-fold increase) in

sik1
-/-

mice fed a high-salt diet as compared to

sik1
-/-

mice on a normal-salt intake. Altogether, this data supports the view that

sik1
-/-

mice fed a high-salt diet develop SNS overactivity. Next, we addressed the question if reducing SNS activity in

sik1
-/-

mice fed a high-salt diet would ameliorate hypertension. For that purpose, the effect of etamicastat, a peripheral reversible DβH inhibitor, was evaluated on the development of high BP upon high-salt diet. Etamicastat treatment (50 mg/kg/day), started prior to high-salt feeding, completely prevented SBP increase in

sik1
-/-

mice fed a high-salt diet (116.8±4.7 mmHg). It is concluded that the SNS is involved in the development of salt-induced hypertension in

sik1
-/-

mice and that the DβH inhibitor etamicastat is able to reduce SNS overactivity and high BP in this mouse model of hypertension.