bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 261: Nitroxyl Anion Induced Vasorelaxation in Pudendal Arteries is Partially Mediated via the Large Conductance Calcium-activated Potassuim Channel
Authors: Labazi Hicham , Wynne Brandi M , Webb R. Clinton
Published: March 2022

Nitroxyl anion (HNO) - a redox congener of nitric oxide (NO) - has been demonstrated to be a potent vasodilator of both conduit and resistance vessels. Previous data from our laboratory have demonstrated that the mechanism of HNO-mediated vasorelaxation varied according to the vascular bed. However, no studies address the role of HNO in erectile function. We have demonstrated that the corpus cavernosum does not exhibit a relaxation response to HNO. However, we have shown that the pudendal artery (PA), which supplies blood flow to the penis, does exhibit a relaxation response to HNO and that ACh-mediated relaxation responses may be partially attributed to HNO as compared to NO. We hypothesized that HNO mediates vasorelaxation in pudendal arteries via activation of potassium (K
+
) channels. PAs were isolated from Sprague-Dawley rats and concentration-response curves (CRC) to an HNO donor: Angeli’s Salt (AS) were performed in phenylephrine contracted vessels (1μM) in the absence and the presence of the calcium (Ca
2+
) -activated K
+
channel inhibitors: TRAM-34 (intermediate Ca
2+
-activated K
+
-channel inhibitor, IK
Ca
, 10 μM) , UCL-1689 (Small conductance calcium-activated potassium channel, SK
Ca
,1 μM), iberiotoxin (large conductance Ca
2+
-activated K
+
-channel inhibitor, BK
Ca
,0.1μM). There were no observed differences in relaxation responses to AS with IK
Ca
and SK
Ca
channel inhibition. In contrast, inhibition of BK
Ca
channels resulted in a rightward shift in the CRC to AS (logEC
50
-4.74±0.11
vs
. -5.26±0.11, p<0.01) with no differences in

R
max

. These data suggest that AS-dependent vasorelaxation in PA is partially mediated via BK
Ca
channel activation.
Abstract 262: Dominant Negative PPARG Influences Atherosclerosis Through Distinct Effects in Vascular Endothelium and Smooth Muscle
Authors: Pelham Christopher , Sigmund Curt
Published: March 2022

Agonists of the nuclear hormone receptor PPARγ inhibit atherosclerosis progression; however, direct actions of PPARγ in the vessel wall during atherosclerosis remain undefined. We reported that transgenic mice expressing dominant negative (DN) PPARγ targeted to the vascular endothelium (E-DN) or smooth muscle (S-DN) on the apolipoprotein E-deficient (ApoE
-/-
) background exhibit increased diet-induced atherosclerotic lesion area in aorta compared to non-transgenic (NT) control mice without changes in plasma cholesterol or triglycerides. Here we show that abdominal aortic rings from standard diet-fed E-DN.ApoE
-/-
mice display impaired endothelium-dependent relaxation in response to Ach (3μM: 44±2% vs 72±1% in NT.ApoE
-/-
; P<0.05) despite normal relaxation to SNP (1μM: 85±1% vs 85±2%; NS) and contraction in response to ET-1 (30nM: 0.84±0.13g vs 0.75±0.08g; NS) and Ang II (10nM: 0.33±0.03g vs 0.33±0.04g; NS). In contrast, abdominal aortic rings from S-DN.ApoE
-/-
mice display impaired nitric oxide-dependent relaxation in response to Ach (3μM: 47±3% vs 71±1% in NT.ApoE
-/-
; P<0.05) and SNP (1μM: 57±3% vs 84±2%; P<0.05) plus enhanced contraction to ET-1 (30nM: 0.98±0.05g vs 0.71±0.12g; P<0.05) and Ang II (10nM: 0.49±0.06g vs 0.36±0.03g; P<0.05). Systolic blood pressure was elevated in E-DN.ApoE
-/-
mice (127±3 vs 112±2 mmHg in NT.ApoE
-/-
; P<0.05) and S-DN.ApoE
-/-
mice (123±1 vs 109±2 mmHg in NT.ApoE
-/-
; P<0.05). Analyzed by real-time qPCR, aorta from E-DN.ApoE
-/-
mice had increased mRNA expression of
Mcp1
(1.68±0.26-fold of NT.ApoE
-/-
; P<0.05) and decreased expression of
Catalase
(0.59±0.13-fold; P<0.05) and
Cd36
(0.69±0.09-fold; P<0.05), while
Cd68
,
Icam1
,
Tnfα
,
Osteopontin
, and
Vcam1
were not changed. Aorta from S-DN.ApoE
-/-
mice had increased mRNA expression of
Mcp1
(1.75±0.23-fold of NT.ApoE
-/-
; P<0.05),
Osteopontin
(2.18±0.13-fold; P<0.05) and
Vcam1
(1.87±0.24-fold; P<0.05) and decreased expression of
Cd36
(0.72±0.08-fold; P<0.05), while
Cd68
,
Icam1
,
Tnfα
, and
Catalase
were not changed. In summary, endothelial- or smooth muscle-specific DN PPARγ induces distinct alterations in vascular function and atherogenic markers. Inhibition of PPARγ function in the vessel wall may contribute to cardiovascular disease.
Abstract 269: Increased Vascular Age in Treated Hypertensive Patients With Elevated Augmentation Index
Authors: Cunha Ana Rosa , Casanova Marcela , Trindade Michelle , D`El-Rei Jenifer , Medeiros Fernanda , Oigman Wille , Neves Mario F
Published: March 2022

Background:
Increased augmentation index has been considered a marker of arterial stiffness and a predictor of cardiac events.

Objective:
To identify clinical and vascular parameters associated with increased augmentation index (Aix) in treated hypertensive patients.

Methods:
A cross-sectional study was carried out to evaluate hypertensive patients on stable drug therapy over at least 4 weeks, both genders, aged 30
-
74 years. Blood pressure (BP), macro and microcirculation parameters were evaluated using high-resolution ultrasonography for brachial flow-mediated dilation (FMD), EndoPat 2000 for peripheral arterial tonometry and Sphygmocor to obtain Aix and other central hemodynamic parameters. The vascular age was also calculated after estimating Framingham risk score for general cardiovascular disease. Patients (n=177) were distributed into three equal groups (n=59 each) according to augmentation index values: 1st tertil (T1) AIx37%.

Results:
The mean age was not different among the groups (55±11 vs 57±10 vs 58±8 years). Although similar diastolic BP was noted in the three groups (87±11 vs 90±13 vs 92±15 mmHg), T3 presented significant increase in brachial systolic BP (139±17 vs 145±17 vs 151±22 mmHg, p<0.006), aortic systolic BP (126±16 vs 138±17 vs 148±24 mmHg, p<0.001), and aortic pulse pressure (41±10 vs 49±14 vs 60±21 mmHg, p<0.001). Aix obtained in microcirculation by EndoPat (14±13 vs 30±19 vs 30±18 %, p<0.001) and vascular age (71±16 vs 77±13 vs 78±12 years, p=0.019) were significantly greater in T2 and T3 comparing to T1. There was no significant differences in endothelial function evaluated by brachial FMD (7.5±5.1 vs 8.9±5.5 vs 9.2±6.7 %) and through reactive hyperemia index by EndoPat (1.9±0.5 vs 2.2±0.7 vs 1.9±0.4 units). Aix was not correlated to age, but there was a significant correlation of Aix with vascular age (r=0.22, p=0.005), brachial systolic BP (r=0.208, p=0.006) and with aortic systolic BP (r=0.404, p<0.001).

Conclusion:
In this population of treated hypertension, increased vascular stiffness characterized by an elevated augmentation index was associated to increased vascular age and raised brachial and aortic systolic BP but not with worsening of endothelial function.
Abstract 275: Age-related Changes in Cardiometabolic and Inflammation Risk Markers in Male SHR Rats
Authors: Pires Nuno M , Igreja Bruno , Wright Lyndon C , Soares-da-Silva Patrío
Published: March 2022

This study examined age-related cardiometabolic and inflammatory changes in spontaneously hypertensive rats (SHR). Aging (from 14 to 59 weeks) was accompanied by increases in systolic blood pressure (166±4 vs 224±5 mmHg, p<0.05), diastolic blood pressure (87±5 vs 132±3 mmHg, p<0.05) and heart rate (376±8 vs 400±5 bpm, p<0.05) leading to ventricular hypertrophy, assessed as ventricular to body weight ratio (3.45±0.0.5 vs 3.66±0.08, p<0.05). Aging SHR showed proteinuria (22±2 vs 46±8 mg/day, p<0.05), increased plasma alanine aminotransferase (39±1 vs 60±6 U/l, p<0.05), cholesterol (1.4±0.1 vs 2.6±0.1 mmol/l, p<0.05), triglycerides (0.4±0.1 vs 0.9±0.1 mmol/l, p<0.05) and glucose levels (154±7 vs 181±5 mg/dl, p<0.05). High levels of plasma C-reactive protein (276±11 vs 447±7 μg/ml, p<0.05) was observed in aged SHR. A multiplex inflammatory cytokine assays was used to profile expression of 23 inflammatory mediators. The measurement of soluble cytokines and other analytes in plasma revealed eleven inflammatory mediators, including IL-1α, IL-6 and INF-γ, significantly increased in aged SHR. In conclusion, SHR aging is accompanied by hypertension establishment and ventricular remodeling. Age-related changes in SHR correlates with increase of cardiometabolic and inflammation risk markers.
Abstract 278: Down-Regulation of Corticosterone Actions Within Nucleus of the Solitary Tract Catecholaminergic Neurons Attenuates Cardiovascular Responses to Stress
Authors: Scheuer Deborah A , Daubert Daisy L , Looney Benjamin M , Clifton Rebekah , Sumners Colin , Sapolsky Robert L , Li Hongwei
Published: March 2022

We previously reported that chronic increases in corticosterone within the dorsal hindbrain, including the Nuclues of the Solitary Tract (NTS), augment the arterial pressure response to restraint stress. This study tested the hypothesis that diminishing actions of corticosterone selectively within NTS catecholaminergic neurons would attenuate the cardiovascular responses to novel and repeated restraint stress. AAV2-based viral vectors were constructed to express either GFP or 11-beta-hydroxysteroid dehydrogenase 2 (HSD2) selectively in catecholaminergic neurons using the PRSx8 promoter. HSD2 degrades corticosterone to an inactive metabolite. Adult male Sprague-Dawley rats were instrumented with telemetry mitters to measure arterial pressure and heart rate. At least 3 weeks later AAV2-PRSx8-HSD2 (n=7) or AAV2-PRSx8-eGFP (n=8) was microinjected bilaterally into the NTS (125 nl/side). After another 3 weeks rats underwent daily restraint stress (60 min per day) for 15 days. Baseline mean arterial pressure was not significantly different in GFP- versus HSD2-transduced rats prior to stress 1 (98±2 and 99±3 mmHg respectively) or stress 15 (96±3 and 100±2 mmHg respectively). During the first restraint stress, the average increase in arterial pressure in response to restraint was significantly (P<0.05) greater in the GFP- compared with the HSD2-transduced rats during the final 10 min of stress (13±1 and 5±2 mmHg respectively). During the last restraint stress the average increase in arterial pressure in response to stress was significantly (P<0.05) greater in the GFP- compared with the HSD2- transduced rats throughout the stress (11±2 and 4±1 mmHg respectively). Baseline heart rate was significantly higher in GFP- versus HSD2- transduced rats prior to stress 1 (340±6 and 317±7 bpm respectively) but not prior to stress 15 (308±6 and 309±9 bpm respectively). HSD2 transduction had no significant effect on the heart rate response to the first stress, but significantly reduced the average heart rate response to the last restraint stress (63±13 and 30±7 bpm for GFP vs HSD2). We conclude that over-expression of HSD2 in NTS catecholaminergic neurons lowers baseline heart rate and attenuates the cardiovascular responses to restraint stress.
Abstract 282: Overexpression of Angiotensin-converting Enzyme 2 Attenuates Tonically Acting Glutamatergic Inputs to the Rostral Ventrolateral Medulla in Hypertensive Rats
Authors: Shen Du , Wang Yangkai , Zhang Ruwen , Wu Zhaotang , Yuan Wenjun , Su Dingfeng , Wang Weizhong
Published: March 2022

The rostral ventrolateral medulla (RVLM) plays a critical role in control of cardiovascular regulation. The increased tonic glutamatergic input to the RVLM contributes to high blood pressure and enhanced sympathetic outflow in hypertensive rats. Recently, it has been reported that overexpression of angiotensin-converting enzyme 2 (ACE2) is capable of lowering high blood pressure in the spontaneously hypertensive rat (SHR). Thus, the aim of this study was to determine the effect of ACE2 gene transfer on tonically acting glutamatergic input to the RVLM in the SHR. It was confirmed that protein expression of ACE2 in the RVLM was increased by an average of 66% in the SHR treated with lentiviral-mediated overexpression of ACE2 (lenti-ACE2) compared with lenti-green fluorescent protein (GFP). A significant reduction in resting blood pressure and the 24-h urinary excretion of norepinephrine was observed in the SHR treated with lenti-ACE2 in the RVLM compared with lenti-GFP. Overexpression of ACE2 significantly reduced the concentration of glutamate (1586±142 μg/L in lenti-GFP versus 621±60 μg/L in lenti-ACE2) from microdialysis-liquid from the RVLM of the SHR. Furthermore, a decrease in resting blood pressure, heart rate, and renal sympathetic nerve activity evoked by microinjection of the glutamate receptor antagonist kynurenic acid (2.7 nmol) into the RVLM was significantly blunted in the SHR treated with lenti-ACE2 compared with lenti-GFP. Based on these data, we suggest that the beneficial effect of ACE2 overexpression in the RVLM on hypertension is associated with reduction in the tonically acting glutamatergic input to the RVLM. (Supported by NSCF: 81170240)
Abstract 293: c-Myc Inactivation by PP2A is Necessary for Dopaminergic Activity in the Proximal Tubule
Authors: Tran Hanh T , Gildea John J , Van Sciver Robert E , Bigler Wang Dora , Felder Robin A
Published: March 2022

We have previously shown that c-Myc regulates the expression of G protein-coupled receptor kinase 4 (GRK4). The inhibition of c-Myc can revert a dopamine-1 receptor (D
1
R) uncoupled renal proximal tubule cell (uRPTC) to normal (nRPTC). The c-Myc inhibitor, 10074-G5 (30 μM, 3 hr), restored coupling of D
1
R mediated adenylyl cyclase stimulation in uRPTCs, causing a significant increase in cAMP levels (16.21% + 0.97; p<0.01; n=47) in response to the dopamine D
1
R/D
5
R agonist, fenoldopam (FEN), than when compared to cells pre-treated with DMSO vehicle (VEH). We have also shown that FEN leads to PP2A activation. Therefore we tested the hypothesis that an inhibitory role of PP2A on c-Myc may play a role in regulating dopaminergic function. PMA (100 nM) was added to either nRPTC or uRPTC for 3hrs. In nRPTC, the total c-Myc from immunoprecipitation increased by 138.7% + 4.1%; p<0.01; n=3 with respect to VEH, while in uRPTC the total c-Myc increased by 214.5% + 11.3%; p<0.01; n=3. The PP2A/Myc association decreased by 46.0% + 1.16%; p<0.05; n=3 with respect VEH in nRPTC and decreased in uRPTC by 46.9% + 8.71%; p<0.05; n=3. These data suggest that c-Myc is activated only when PP2A binding is low. Addition of FEN (3 hrs) decreased the total c-Myc in nRPTC by 22.6% + 9.1%; p=0.06; n=3 but not in uRPTC. In nRPTC, the PP2A/Myc association increased by 56.6% + 15.1%; p <0.05; n=3 when compared to VEH, but showed no response in uRPTC. Addition of natriuretic peptide angiotensin-III (Ang III, 10 nM) decreased the expression of c-Myc by 29.4% + 16.8%; p<0.05; n=3, but showed no response in uRPTC. The PP2A/Myc binding increased by 109.3% + 30.1%; p<0.05; n=3 in nRPTC, with no increase in uRPTC. Here we show for the first time in nRPTC that both FEN and Ang III decrease total c-Myc while increasing PP2A/Myc co-immunoprecipitation, leading to inactivation of c-Myc. In uRPTC, no significant change in the total c-Myc or the association of PP2A/Myc was detected in response to FEN or Ang III. This suggests that both a lack of expressional control of c-Myc, in addition to a lack of inhibition of c-Myc by PP2A in uRPTC, may have a role in the etiology of D
1
R uncoupling and human renal proximal tubule-related hypertension. Therefore, c-Myc inhibitors may provide a new approach for treating hypertension.
Abstract 305: Association Between CYP4A11 Variant and Response to Spironolactone, but Not ENac Inhibitor Amiloride
Authors: Brown Nancy J , Eckert George J , Tu Wanzhu , Pratt J H
Published: March 2022

CYP4A11
polymorphisms have been associated with hypertension (rs1126742) and CAD (rs3890011) in humans. In mice, deficiency of the
Cyp4a
gene causes ENaC dysfunction and salt-sensitive hypertension that is abolished by the ENaC inhibitor amiloride. We examined the relationship between genotypes in
CYP4A11
and the BP response to amiloride, spironolactone, both drugs, and placebo in 83 hypertensive African American participants in a previously published randomized clinical trial. Genotype frequencies for rs3890011 were CC:GC:GG=28:35:20. The frequency of the loss-of-function variant at rs1126742 was similar to that previously reported (CC:TC:TT=7:31:45), but too small to permit further analysis. There was no difference in baseline SBP (overall mean±SE 142±1) or number of medications (2.3±0.1) among genotype groups. RS3890011 C allele was associated with an attenuated SBP responses to spironolactone (7.3±4.5 in CC versus -9.3±2.6 in GC or GG in the spironolactone only group, p=0.002; -2.7±3.3 in CC versus -12.8±2.3 in GC or GG in subjects receiving spironolactone alone or in combination, p= 0.014, Figure) and similar for DBP (5.0±2.9 in CC versus -6.0±1.7 in GC or GG with spironolactone only, p=0.002; -1.2±2.0 in CC versus -6.3±1.4 in GC or CC in those receiving any spironolactone, p=0.043). Spironolactone reduced BP only in carriers of the rs3890011 G allele. In contrast, there was no difference among genotype groups in the BP response to ENaC inhibitor amiloride. Variation in
CYP4A11
influences the response to spironolactone but not amiloride; these data are consistent with studies in genetically modified mice suggesting that a product of CYP4A11 modifies ENaC function.
Abstract 328: Left Ventricular Diastolic Dysfunction, Regardless of Classification is Associated with increased Muscle Sympathetic Nerve Activity in Hypertensive Patients.
Authors: Souza Silvia B , Rocha Juraci A , Cuoco Marco A , Bortolotto Luiz A , Krieger Eduardo M , Consolim-Colombo Fernanda M
Published: March 2022

Background:
The presence of asymptomatic left ventricular diastolic dysfunction (LVDD) in hypertensive patients can be associated with the development of cardiac events. The increase in the sympathetic activity is considered one of the mechanisms that predisposes to this outcome. The aim of this study was to investigate whether there is an increase of MSNA in the presence of LVDD in hypertensive patients under therapy, and whether it influences or not the different classes of diastolic dysfunction in the MSNA. Method: After analyzing left ventricular function by echoDopplercardiography exam, 45 hypertensive patients were allocated into 3 groups: normal function (H, n=15); impaired relaxation (H-IR, n=15) and pseudonormal or restrictive (H-P/R, n=15). An age, sex and body mass index- matched control group of normotensive volunteers (N, n=14) was included. In all groups, muscle nerve sympathetic activity (MNSA) by microneuragraphy, and blood pressure by FINOMETER® were evaluated in supine rest position. Evaluation statistics: ANOVA and Fisher’s Exact.

Results:
Blood pressure and antihypertensive drugs were similar among hypertensive groups. Both H-IR and H-P/R had a similar MSNA (33±1 and 32±1 bursts/min respectively) which was significantly higher compared to H and N groups (26±3 and15±2 bursts/min respectively).

Conclusions:
1. Increased MSNA in treated hypertensive patients exhibiting LVDD can be detected. 2. The increased MSNA is not influenced by different classes of LVDD. 3.The clinical implication of this finding should be further evaluated, considering the worse prognosis already established when LVDD is present.
Abstract 33: Participation of Prostaglandin E2 and EP4 Receptors in Connecting Tubule Glomerular Feedback (CTGF).
Authors: Ren YiLin , D'Ambrosio Martin A , Wang Hong , Garvin Jeffrey L , Carretero Oscar A
Published: March 2022

The afferent arteriole (Af-Art) accounts for most of renal vasculature resistance, thus controlling glomerular filtration rate. The connecting tubule (CNT) is in close contact with the Af-Art. Connecting tubule glomerular feedback (CTGF) is a mechanism in which Na reabsorption in the CNT causes Af-Art dilation. We have shown that CTGF is mediated by eicosanoids, including prostaglandins and epoxyeicosatrienoic acids (EETs), however their exact source and nature remain unknown. Here we tested the hypothesis that during CTGF the CNT releases prostaglandin E
2
(PGE
2
), which binds EP4 receptors and dilates the Af-Art. Rabbit Af-Arts with the adherent CNT intact were microdissected and perfused. The Af-Arts were preconstricted with norepinephrine. CTGF was elicited by increasing luminal NaCl in the CNT from 10 to 80 mM. To identify the source of the vasodilator prostaglandins we first disrupted the vascular endothelium by adding anti-Factor-VIII antibody to the Af-Art lumen. This did not affect CTGF (control: 7.9±0.9, endothelium disrupted: 8.6±0.6 μm, n=7). We then added arachidonic acid (AA) to the lumen of the CNT while maintaining zero NaCl in the perfusate, and found that AA caused dose-dependent dilation of the attached Af-Art (from 7.9±2.2 to 15.5±1.3 μm,
P
<0.01, n=4). This cannot be explained by AA diffusing to the Af-Art because AA is a vasoconstrictor for the Af-Art. Taken together, these data indicate that CTGF-induced vasorelaxation is endothelium-independent, and that prostaglandins originate from the CNT. To study which specific prostaglandin and receptor mediate CTGF, we added the EP4 receptor blocker L161982 (10 μM) to the bath in the presence of the EET synthesis inhibitor MS-PPOH. EP4 receptor blockade abolished CTGF (control: 8.5±0.9, MS-PPOH+L161982: 0.8±0.4μm,
P
<0.001, n=6). To confirm these results we used a different, specific EP4 blocker, ONO-AE-208 (100 nM), which also abolished CTGF (control: 9.4±0.5, MS-PPOH+ONO-AE-208: -0.6±0.2 μm,
P
<0.001, n=6). We conclude that increasing luminal Na stimulates release of prostaglandin E
2
from the CNT, which acts on EP4 on the Af-Art inducing endothelium-independent dilation during CTGF. Our studies provide a better understanding of the mechanisms by which CTGF controls Af-Art resistance.
Abstract 343: EGF Deficiency in the Renal Cortex Contributes to Salt-sensitive Hypertension via Upregulation of ENaC Activity
Authors: Pavlov Tengis S , Levchenko Vladislav , O’Connor Paul M , Ilatovskaya Daria V , Palygin Oleg , Mori Takefumi , Mattson David L , Sorokin Andrey , Lombard Julian H , Cowley Allen W , Staruschenko Alexander
Published: March 2022

Dahl salt-sensitive (SS) rats fed a high salt diet exhibit increased blood pressure and renal damage. Epithelial sodium channels (ENaC) in the aldosterone-sensitive distal nephron (ASDN) are responsible for the fine tuning of Na
+
absorption in the kidney and are regulated by various stimuli including hormones and growth factors. Members of the epidermal growth factor (EGF) family are important for maintaining transepithelial Na
+
transport. A combination of electrophysiological, immunohistochemical, biochemical
in vitro
and chronic
in vivo
studies were used to provide mechanistic insights into ENaC regulation by EGF and how changes in this pathway may contribute to salt-induced hypertension in SS rats. It was found that the ENaC inhibitor benzamil, administered through the drinking water (15 mg/L), significantly reduced the MAP from 173.3±15.0 to 127.0±10.1 mm Hg. ENaC expression (as measured by Western blotting and immunohistochemistry) and activity (single channel analysis in freshly isolated split open ASDN) were significantly enhanced in SS rats fed a high salt diet compared to SS rats fed a low salt diet and consomic SS.13
BN
rats fed a high salt chow. Servo-controlled measurements in SS rats also revealed increased ENaC expression in the uncontrolled kidneys compared to controlled left kidneys. As assessed by ELISA assay, EGF level in the kidney cortex of SS rats fed a high salt diet was lower compared to rats on a low salt diet. To directly evaluate the role of EGF in the development of hypertension and its effect on ENaC activity, EGF (10 μg/h/kg) was intravenously infused and blood pressure was continuously monitored. Chronic infusion of EGF decreased ENaC activity (

NP
o

; from 1.5±0.2 to 0.4±0.1), prevented the development of hypertension (from 184.0±8.9 to 149.0±9.6 mm Hg) and attenuated renal glomerular and tubular damage. Thus, in controversy to existing dogma, we provide an evidence of a critical role of ENaC in the ASDN in the development of salt-sensitive hypertension. Furthermore, these findings indicate that EGF deficiency in the renal cortex contributes to salt-sensitive hypertension and that
in vivo
EGF infusion prevents development of hypertension and kidney damage via downregulation of ENaC.
Abstract 347: The Role of Matrix Metalloproteinase-2 During the Development of Hypertension-induced Renal Injury in Dahl Salt-sensitive Rats.
Authors: Williams Jan M , Slaughter Tiffani N , Paige Adrienne , Chen Chun Cheng , Fan Fan , Guerts Aron M , Jacob Howard J , Roman Richard J
Published: March 2022

We recently reported that the progression of hypertension-induced renal injury in Dahl salt-sensitive (SS) rats was associated with increased renal expression of matrix metalloproteinase-2 (MMP-2) protein. Moreover, chronic treatment with a non-selective MMP inhibitor reversed the progression of renal injury in SS rats fed a high salt (HS) diet. In the present study, we created an 8 base pair frame-shift deletion in exon 7 of the MMP-2 gene within the genetic background of the SS rat (MMP-2 ZN KO strain) using
Zinc-finger
nucleases to study MMP-2 during the development of hypertension-induced renal injury in SS rats fed a HS diet containing 8.0% NaCl. MAP (measured by telemetry) rose from 112±3 to 166±10 mmHg in SS rats (n=6) fed a HS diet for 4 weeks. In contrast, MAP only increased to 140±4 mmHg in MMP-2 ZN KO rats (n=10). Proteinuria rose from 82±9 to 267±18 in SS rats fed a HS diet for 4 weeks
vs.
24±2 to 139±11 mg/day in the MMP-2 ZN KO strain. MMP-2 protein and activity (measured by zymography) were significantly higher in the renal cortex of SS rats fed a HS diet when compared to the levels observed in SS rats on a low salt (LS) diet containing 0.3% NaCl. We detected very low levels of MMP-2 protein expression and activity in the renal cortex and glomeruli of MMP-2 ZN KO rats relative to that seen in SS rats fed either a LS or HS diet. The kidneys of SS rats fed a HS diet for 4 weeks exhibited severe glomeruloslcerosis and renal fibrosis and the degree of renal injury was markedly reduced in the MMP-2 ZN KO strain. These results indicate that upregulation of MMP-2 contributes to the development of hypertension-induced renal injury in SS rats fed a HS diet and suggest that specific MMP-2 inhibitors may be a new therapeutic target to slow the progression of chronic kidney disease in hypertensive and diabetic patients.
Abstract 349: Comparison of Systolic, Diastolic, and Pulse Pressure, and Blood Pressure Variability, as Predictors of Kidney Function Loss in Persons With Chronic Kidney Disease in the Chronic Renal Insufficiency Cohort
Authors: Townsend Raymond R , Anderson Amanda Hyre , Feldman Harold I , Kallem Radakrishna , Yang Wei Peter , Kusek John , Lash James P , Rahman Mahboob , Chertow Glenn M , Steigerwalt Susan , Wright Jackson , Miller Edgar , Charleston Jeanne
Published: March 2022

Blood pressure (BP) predicts kidney function decline in CKD. It’’s unclear if systolic (SBP), diastolic (DBP), or pulse pressure (PP) is the strongest predictor, or if BP variability is a determinant. In a prospective study of CKD we examined the association of 3 brachial BP measures on a composite kidney function outcome: end-stage kidney disease (ESKD) or a halving in the estimated GFR compared to baseline. We also examined whether the amount of variability within the 3 BP measurements at baseline predicted the outcome. CRIC participants (∼half DM; ∼half M; eGFR 44.8(16.8) mL/min/1.73 m
2
) had 3 seated brachial BP at study baseline. Mean BP [mmHg(SD)] and PP were 128(22)/72(13) and 57(19). Mean BP variability, as the SD of SBP (SBPSD) at baseline was 3.3(2.5). After 3.7 years, 766 participants had a halving in eGFR from baseline or ESKD. We modeled the time to the event with Cox proportional hazards regression. For comparability across different BP measures, results were expressed per 1 SD difference in SBP, DBP, and PP; adjusting for eGFR, proteinuria, age, gender, race, clinical site and diabetes(Y/N) in the 3379 participants with complete data. Death was a censoring event. Akaike Information Criterion (AIC; lower values better) evaluated overall model fit characteristics. The same regression parameters were used to model CKD associated with SBPSD, including the mean of the SBP at baseline as a covariate in this analysis.

SBP was the strongest BP predictor of loss of kidney function. Variability of baseline SBP also predicted loss of kidney function, independent of level of SBP at baseline, but this association was considerably weaker than those observed for other BP measures.
Abstract 350: Renal Dysfunction Associated With Incident Hypertension According to Blood Pressure Categories in a Non-hypertensive Population in the Suita Study: An Urban Cohort Study
Authors: Kokubo Yoshihiro , Watanabe Makoto , Nakamura Satoko , Kawanishi Katsuyuki , Miyamoto Yoshihiro
Published: March 2022

Background and purpose:
No studies have examined the influence of decreased glomerular filtration rate (GFR) on incident hypertension (HT) according to blood pressure (BP) categories among a healthy cohort. We assessed the relationships between renal dysfunction and incident HT according to BP categories in a Japanese urban cohort.

Methods:
A total of 4,751 participants (30 to 79 years old without HT at baseline) in the Suita Study were prospectively followed up for incident HT. BP categories were defined by the ESH-ESC in 2007 criteria from an every 2 years health examination by well-trained physicians. Systolic and diastolic BPs (SBP and DBP, respectively) were taken as the average of the 2nd and 3rd measurements. Chronic kidney disease (CKD) was defined as GFR <60 mL/min/1.73 m
2
, which was estimated using the MDRD equation modified by the Japanese coefficient. Urinary protein was determined by using a dipstick method. Urinary protein results of "+-" or more grade were defined as proteinuria. The Hazard ratios (HRs) and 95% confidence intervals (CIs) of incident HT associated with CKD and proteinuria were calculated by using the Cox proportional hazard models adjusted for age, sex, diabetes, dyslipidemia, body mass index, exercise, smoking, and excessive drinking.

Results:
During the 7.2 years of mean follow-up, 21, 63, and 137 cases of incident HT per 1000 person-years were observed in the optimal, normal, and high-normal BP (HNBP) categories, respectively. The adjusted HRs (95% CIs) for incident HT were 1.2 (1.1-1.5) in the CKD group, compared with the non-CKD group. The HRs (95% CIs) were 1.2 (1.1-1.4) and 1.7 (1.4-2.1) in the urinary protein "+-" and "1+" or more groups, respectively. The HRs (95% CIs) of the non-CKD and proteinuria, CKD and non-proteinuria, and CKD and proteinuria groups were 1.5 (1.2-2.0), 0.9 (0.6-1.3), and 1.8 (1.0-3.1) in the normal BP and 1.3 (1.1-1.6), 1.3 (1.0-1.8), and 1.8 (1.2-2.6) in the HNBP categories, respectively, compared with the same BP categories of the non-CKD and non-proteinuria group.

Conclusions:
CKD and urinary protein are risk factors for incident HT in the non-hypertensive population. This study emphasizes the importance of urinary protein in prehypertension for the primary prevention of HT, even if they have non-CKD.
Abstract 351: Temperature Dependency of Blood Pressure and Heart Rate in Norepinephrine-Deficient Mice
Authors: Garland Emily M , Finney Charlene , Appalsamy Martin , Mai Tu , Diedrich André , Robertson David
Published: March 2022

Dopamine beta-hydroxylase (Dbh) knockout mice lack the sympathetic neurotransmitter, norepinephrine, but have intact sympathetic neurons and adrenergic receptors. These mice offer an excellent model for assessing the role of the sympathetic nervous system in physiological and pathophysiological processes. Mouse blood pressure (BP) and heart rate (HR) increase as ambient temperature drops below the thermoneutral temperature (30° C), apparently due to sympathetic nervous system activation. The present study tested the hypothesis that BP and HR are not sensitive to changes in ambient temperature in Dbh knockout (KO) mice lacking sympathetic function. BP, HR and activity data were obtained at least one week after BP telemeters were implanted in the carotid artery. Eight Dbh wild type (WT) and 7 KO mice were initially maintained at 23° C, singly housed in temperature-controlled brooders with a 12-hour light-dark cycle. With 3° C temperature changes every 3-4 days, chambers were warmed to 32° C, followed by cooling back to 23° C. Twenty-four hour averaged BP and HR were significantly lower in KO than WT mice at all temperatures, although activity levels were no different. HR, averaged over 24hr, were significantly higher at 23° C after cooling from 32° C for both genotypes (mean±SD; KO: 376±24 to 462±53 beats/min, P=0.016; WT: 411±22 to 542±46 beats/min, P=0.008). In contrast, BP increased with the drop in temperature only in the WT mice (KO: 83±7 to 87±10 mmHg, P=0.109; WT: 94±9 to 107±9 mmHg, P=0.008), and BP differences between 32° and 23° C were greater in WT mice (delta BP
23
°
-32
°
averaged 12.2±5.4 mmHg for WT and 4.2±5.0 mmHg for KO, P=0.011). Activity did not change as ambient temperature was lowered. These data indicate that effects of ambient temperature on HR occur independently of sympathetic activation, but our results confirm that an intact sympathetic nervous system is required for the pressor effect of a cooler environmental temperature.
Abstract 354: Mitochondrial Hyper-function in Renal Proximal Tubule Cells from Young Spontaneously Hypertensive Rats
Authors: Lee Hewang , Abe Yoshifusa , Lee Icksoo , Shrivastav Shashi , Hüttemann Maik , Felder Robin A , Jose Pedro A , Kopp Jeffrey B
Published: March 2022

Recent evidence suggests that mitochondria play roles in the pathogenesis of hypertension. However, it is not clear whether mitochondrial dysfunction precedes or follows the development of hypertension. We hypothesize that mitochondrial bioenergetics is altered in renal proximal tubule (RPT) cells from spontaneously hypertensive rats (SHRs) and this alteration may occur prior to the development of sustained hypertension. Using immortalized RPT cells from 4-8 week old normotensive Wistar-Kyoto (WKY) and SHRs, we measured oxygen consumption rate (OCR) and compared the parameters of cellular bioenergetic in these RPT cells. Basal OCR was significantly higher in RPT cells from SHRs than WKY rats (245.5±31.9 vs. 154.4±43.7 pmol/min per 20,000 cells, mean±SD, P<0.001);the ATP synthesis-coupled OCR (65.9±3.3% vs. 55.0±6.6%, of basal OCR, P<0.001), maximum respiration (609.0±120.4 vs. 215.7±65.5 pmol/min per 20,000 cells, P<0.001) and reserve respiration (147.8±39.0% vs, 40.0±15.4% of basal OCR P<0.001) were also significantly higher in RPT cells from SHRs than WKY rats. In contrast, proton leak-linked OCR (15.4±3.6% vs. 19.2±5.9% of basal OCR, P<0.05) was lower in RPT cells from SHRs than WKY rats, and non-mitochondrial OCRs were similar (45.2±7.0 vs. 41.3±19.8 pmol/min per 20,000 cells, P>0.05). In support of these observations, adenosine triphosphate (ATP) levels were significantly higher in RPT cells from SHRs than WKY rats. Taken together, the observed bioenergetic alterations may contribute to the early mitochondrial dysfunction in RPT cells from SHRs prior to the onset of sustained hypertension.
Abstract 355: MYH9 E1841K Mutation Enhances Susceptibility to Angiotensin-II Induced Proteinuria and Podocyte Injury That is Ameliorated by AT1 Receptor Inhibition.
Authors: Cechova Sylvia , Rudy Christine K , Chan Fang R , Kelly Michael J , Le Thu H
Published: March 2022

The
MYH9
gene encodes the nonmuscle myosin heavy chain IIA. Recent genome-wide association studies (GWAS) demonstrated that intronic variants of the
MYH9
gene are associated with non-diabetic kidney disease and hypertension-attributed ESRD in African-Americans. However, the causal functional variants of
MYH9
have remained elusive. Rare autosomal-dominant missense mutations in MYH9 cause macrothrombocytopenia, and are occasionally associated with development of nephropathy. The E1841K mutation is the most common
MYH9
missense mutation and has been associated with nephropathy in some carriers. To determine the role of the
MYH9
E1841K mutation in the development of kidney disease, we studied a mouse line carrying the E1841K mutation. On a mixed genetic background (129S6 and C57BL/6), there was no difference in systolic blood pressure (SBP) between wild-type (WT, -/-) and mice with the E1841K mutation (designated as
E1841K (+)
: +/- and/or +/+) at baseline or after high salt diet (HSD,6 % NaCl). There was also no difference in albumin/creatinine (A/C ratio) at baseline or after HSD between groups. We next assessed the effects of the E1841K mutation in response to angiotensin II (Ang II) infusion @1000 ng/kg/min for 28 days via mini-osmotic pump. Despite similar increases in SBP after Ang II (WT 197 ± 4 mm Hg, n=15;
E1841K +/-
197 ± 3 mm Hg, n=9;
E1841K +/+
199 ± 5 mm Hg; n=6),
E1841K(+)
mice displayed severe proteinuria in a dose-dependent manner (A/C ratio (ug/mg): WT 806±94,
E1841K +/-
1341±293,
E1841K +/+
2209± 366, p=0.001, ANOVA ). By light microscopy, WT mice had only mild mesangial expansion, whereas
E1841K +/+
mice had focal segmental glomerulosclerosis and large amounts of proteinaceous casts. By electron microscopy, WT mice had intact podocytes, whereas,
E1841K +/+
mice displayed severe podocyte foot process effacement/fusion. Treatment of
E1841K (+)
mice with candesartan, an angiotensin type 1 receptor (AT
1
R) inhibitor, significantly prevented Ang II-induced hypertension and proteinuria (
E1841K (+)
(n=6; +/- (n=4) and +/+ (n=2)): SBP = 138 ± 4 mm Hg, A/C ratio = 111 ± 47 ug/mg; p ≤ 0.0001 vs. untreated), and ameliorated podocyte foot process effacement in
E1841K +/+
mice. In conclusion, the
MYH9
E1841K mutation predisposes to AT
1
R mediated podocyte injury.
Abstract 364: Nrf2-PPAR gamma Signaling Suppresses Exaggerated Renal Tubular AT1R Gene Expression and Function and Mitigates Hypertension During Oxidative Stress
Authors: Banday Anees Ahamd , Lokhandwala Mustafa F
Published: March 2022

Oxidative stress plays an important role in the development of hypertension. Interestingly, angiotensin type 1 receptor (AT1R) function, a major contributor to the etiology of hypertension, is positively regulated by oxidative stress. It is shown that antioxidant treatment can reduce blood pressure (BP) and redox-sensitive transcription factor Nrf2 could provide important clues for antihypertensive properties of antioxidants. Here in we investigated how induction of renal proximal tubular Nrf2 in response to antioxidants will activate PPAR gamma (a pleiotropic nuclear receptor whose ligands exhibit BP reducing effects) which in turn will normalize AT1R expression and reduce BP. Wild type (WT) and renal proximal tubular specific Nrf2 knockout (KO) mice were treated with 10 mM L-buthionine-sulfoximine (BSO, a pro-oxidant) without and with sulforaphane (S, an antioxidant; 5 μM daily by gavage) for 5 wks. BSO-treatment increased oxidative stress, AT1R mRNA, AT1R expression and Ang II-induced activation of renal sodium transporters and these animals exhibited high BP (mm Hg, WT: 102±3, WT-BSO: 123±4, Nrf2 KO: 98±2). However, the onset and severity of hypertension (Nrf2-KO-BSO: 136±4) as well as oxidative stress and AT1R function was more robust in BSO-treated Nrf2 KO compared to WT mice. More importantly, sulforaphane activated Nrf2, reduced oxidative stress, normalized proximal tubular AT1R function and reduced BP (WT-S: 99±5, WT-BSO+S: 106±6) in BSO-treated WT mice but failed to mitigate AT1R function or reduce BP (Nrf2 KO-S: 97±4, Nrf2 KO-BSO+S: 130±3) in Nrf2 KO mice. Further studies showed that BSO-induced hypertension is also more severe in renal proximal tubular specific PPAR gamma KO mice and sulforaphane fails to protect PPAR gamma KO mice from hypertension. Experiments in human renal proximal tubular cells indicated that 1) sulforaphane, via Nrf2 activation, reduced oxidant-induced AT1R upregulation and 2) Nrf2 dependent PPAR gamma activation suppresses AT1R gene transcription. These findings suggest that antioxidants via Nrf2 activation reduce oxidative stress, normalize AT1R signaling and prevent hypertension during oxidative stress. More importantly, Nrf2 induction activates PPAR gamma which suppresses AT1R gene transcription.
Abstract 367: A Nox4 Inhibitor, GKT 137831, Decreases Aldosterone Synthesis, Adiposity and Vascular Contractility in a Murine Model of Diabetes.
Authors: Montezano Augusto C , Nguyen Dinh Cat Aurelie , Antunes Tayze T , Callera Glaucia E , Briet Marie , He Ying , Page Patrick , Szyndralewiez Cedric , Heitz Freddy , Cooper Mark E , Touyz Rhian M
Published: March 2022

Nox4 is expressed in many cell types including vascular cells and adipocytes. Expression of Nox4 is increased in cardiovascular diseases, such as hypertension and diabetes. Despite the advancement in the understanding of Nox4 biochemistry and its mechanisms of regulation, the pathophysiological role of Nox4 remains elusive and controversial. We recently demonstrated that adipocytes produce aldosterone, an effect that was exacerbated in diabetes and contributed to vascular dysfunction. Here we postulated that Nox4 plays a role on adipocyte-derived aldosterone production, leading to vascular dysfunction, during diabetes. Studies were performed in db/m (control) and db/db mice (a murine model of diabetes). Control and db/db mice were divided into 3 groups: Group 1, no treatment, Group 2, treatment with 20 mg (low dose) and Group 3, 60 mg (high dose) of GKT137831 (GKT) (Nox4 inhibitor) for 16 weeks. Blood pressure was measured by tail-cuff. Aldosterone (aldo) levels were measured in plasma and cell culture media from mature adipocytes extracted from all groups. Mesenteric arteries were obtained from all groups to study vascular responses to acetylcholine, a nitric oxide donor (DEA-NO) and norepinephrine (NE) by myography. Body weight was increased in db/db (61.8g ± 0.95) versus control (33.5g ± 0.72) mice, p<0.05. GKT treatment did not alter body weight in db/m or db/db mice. Blood pressure levels were similar in all groups. Epididymal adiposity was increased in db/db mice (0.098g ± 0.16 vs. 0.067g ± 0.15, p<0.05). Aldo levels were increased in plasma (pg/mL: 518 ± 67 vs. 272 ± 30, p<0.05) and media from mature adipocytes (pg/mL/μg RNA: 1964 ± 213 vs. 388 ± 55, p<0.001). GKT treatment (high dose) decreased adiposity (0.07g ± 0.05) and aldo levels in plasma (390 ± 41 pg/mL) and media from mature adipocytes (577 ± 91 pg/mL), p<0.01. Endothelial dysfunction observed in db/db mice was not affected by GKT treatment. However, GKT treatment induced a decrease on NE-induced contraction in mesenteric arteries from db/db mice (pD2 values: db/db vs. db/db high dose, 6.4±0.1 vs. 5.6 ± 0.1, p<0.001). Our findings suggest a role for Nox4 in adipocyte-derived aldo production and vascular reactivity in db/d mice. Such Nox4-related processes may be important in obesity-diabetes.
Abstract 373: Impacts of Apocynin and Allopurinol on Exercise Training-increased Nitric Oxide Synthase Activity and Expression in the Kidney of SHR and WKY
Authors: Cao Pengyu , Ito Osamu , Ito Daisuke , Sakuyama Akihiro , Rong Rong , Muroya Yoshikazu , Sakata Yoshiko , Mori Nobuyoshi , Kohzuki Masahiro
Published: March 2022

It has been recently reported that the exercise training (Ex) increases nitric oxide (NO) production and NO synthase (NOS) expression not only in vasculatures but also in the kidney of spontaneously hypertensive rats (SHR) with the reduction of systemic blood pressure. To clarify the mechanism of the Ex-increased NOS expression, the impacts of inhibitors of NADPH oxidase and xanthine oxidase on the Ex-increased NOS activity and expression were examined in SHRs and Wistar-Kyoto rats (WKY). Five week-old, male SHR or WKY were trained with treadmill running. Apocynin (2 mmol/L in drinking water), an inhibitor of NADPH oxidase or allopurinol (1.5 mmol/L in drinking water), an inhibitor of xanthine oxidase was given for drug treatments. After 8 weeks, H2O2 and NO2/NO3 (NOx) in plasma and urine were measured. The NOS activity and expression were examined in the kidney cortex, the outer medulla, the inner medulla and thoracic aorta. In both SHR and WKY, the Ex significantly increased H2O2 and NOx in plasma and urine, NOS activity and endothelial and neuronal NOS (eNOS and nNOS) expressions in the kidney cortex, the outer medulla, the inner medulla and thoracic aorta (p<0.01 in each). Apocynin significantly decreased basal levels of H2O2 and NOx in plasma and urine, NOS activity and eNOS and nNOS expressions (p<0.01 in each) in the kidney sections and aorta of SHR, but did not affect all parameters in those of WKY. Apocynin blocked the Ex-induced changes of H2O2 and NOx levels and renal and aortic NOS activity and expression in WKY and aortic NOS activity and expression in SHR, but it did not block the Ex-induced changes of H2O2 and NOx levels or renal NOS activity and expression in SHR. In contrast to apocynin, allopurinol did not affect basal levels of all these parameters in SHR or WKY. Allopurinol blocked the Ex-induced changes of H2O2 and NOx levels and renal NOS expression in SHR but did not block the Ex- induced changes of aortic NOS expression in SHR or all parameters in WKY. These results indicate that the Ex-increased NOS activity and expression in aorta were mediated through NADPH oxidase in both SHR and WKY. The Ex-induced NOS activity and expression in the kidney were mediated through NADPH oxidase in WKY but through xanthine oxidase in SHR.
Abstract 382: Studies on the Effect of ACE2 Inhibitors on ACE2-driven Angiotensin Peptides Metabolism in Plasma Using RAS Fingerprinting
Authors: Wysocki Jan , Ye Minghao , Poglitsch Marko , Schuster Manfred , Batlle Daniel
Published: March 2022

There are species differences between the currently used ACE2 inhibitors (MLN-4760 and DX600) that could be critically relevant to the proper interpretation of studies with these compounds. The effect of these inhibitors on ACE2-driven angiotensin peptides formation and degradation has not yet been studied. Accordingly, we examined the ability of MLN-4760 and two conformational variants of DX600 [a linear (L) and a cyclic (C) form] to inhibit human (h) and mouse (m) recombinant (r) ACE2 using a novel ex vivo LC-MS/MS based approach to concomitantly measure several peptides within the RAS system in plasma. Both mrACE2 and hrACE2 (5ug/mL) when added to blood plasma caused a complete disappearance of Ang II and lead to the formation of Ang-(1-7). As a result of the increase in Ang-(1-7) formation, both mrACE2 and hrACE2 lead also to the formation of Ang-(1-5). This reaction was likely driven by ACE which, like other peptidases, is present in plasma ex vivo assay. The effects of hrACE2 and mrACE2 on the above mentioned Ang peptides were inhibited by MLN-4760 at 10-5M and also at lower concentrations (10-6M and 10-7M). Both, L-DX600 and C-DX600 inhibited the effect of hrACE2 on Ang-(1-7) and Ang-(1-5) formation and this effect was also dose-dependent. By contrast, neither L-DX600 nor C-DX600, even at a high concentration (10-5M), prevented the changes on Ang peptides effected by mrACE2. hrACE2 decreased Ang I levels by 60% whereas mrACE2 did it only marginally. Of note, hrACE2 resulted in the formation of Ang-(1-9) from Ang I whereas mrACE2 did not. In conclusion, there are major differences between the known specific inhibitors of ACE2 between hrACE2 and mrACE2. MLN-4760 inhibits both hrACE2 and mrACE2 whereas DX600 inhibits hrACE2 preferentially. DX600 does not prevent mrACE2-driven formation of Ang-(1-7) from Ang II. mrACE2 effectively degrades Ang II forming Ang-(1-7) whereas it has no effect on the formation of Ang-(1-9) from Ang I. RAS “fingerprinting” is a powerful novel approach to simultaneously evaluate quantitatively several angiotensin peptides in plasma and can be used to demonstrate profound differences on the effect of available ACE2 inhibitors on the formation of various angiotensin peptides driven by human and mouse ACE2.