AT1R mRNA Research Articles

O48 CHRONIC EXPOSURE TO POLLUTED URBAN AIR ALTERS MOUSE SYSTEMIC AND TISSUE-SPECIFIC COMPONENTS OF THE RENIN-ANGIOTENSIN SYSTEM (RAS)

Background and Objective: Chronic exposure to air pollution induces pulmonary, cardiovascular, and renal diseases. In the current study, we investigated the hypothesis that postulates that a dysregulation of the RAS may contribute to these pathologies. Methods: Male Balb/C mice were exposed to urban air from Buenos Aires City, in whole-body exposure chambers for 14 weeks. Control animals were exposed to filtered air. The levels of main RAS components including angiotensin-converting enzyme (ACE) and ACE2; AT1, AT2, and Mas receptors (R) were determined in mouse lungs, kidneys, and heart, through western blotting (WB) and RT-qPCR, respectively. The tissue content of cytokine mARN was determined by RT-qPCR. The tissue content of nitrotyrosine and 4-hydroxynonenal (4-HNE) levels, were determined by WB. Circulating angiotensin (Ang) II levels were determined by radioimmunoassay. Systolic blood pressure was measured by the tail-cuff method. Results: Exposure to air pollution resulted in a) Increased levels of ACE and AT1R mRNA in the lungs and augmented the abundance markers of inflammation (TNF-α, IL-6, and IL-1β) and oxidative stress (nitrotyrosine and 4-HNE) in this tissue, b) increased levels of MasR mRNA (all analyzed tissues) and MasR protein levels (lungs and heart), c) upregulated mRNA and protein abundance of cardiac and renal AT2Rs, and d) Increased levels of circulating Ang II and e) augmented systolic blood pressure. Conclusions: Our findings indicate that chronic exposure to air pollution alters the expression of both tissue and systemic components of the RAS. Since both AT2R and MasRs have been shown to participate in tissue-repair mechanisms, the upregulation of these receptors detected in mouse lungs, heart, and kidney after chronic exposure to polluted urban air could represent a mechanism of tissue protection against damage induced by air pollution.

Abstract P170: Angiotensin II Receptor Associated Protein Expression Within the PVN of Spontaneously Hypertensive Rats

Increased levels of circulating Angiotensin II (Ang II) have been linked to the development of neurogenic hypertension and elevated sympathetic activity, primarily through the Angiotensin II Receptor 1 (AT1R). Angiotensin II Receptor Associated Protein (ATRAP) is a transmembrane protein that regulates Ang II signaling by promoting the internalization of AT1R. Studies have demonstrated that in a model of Ang II-induced hypertension, enhancing ATRAP expression in the paraventricular nucleus of the hypothalamus (PVN) via Lentivirus infection reduced high blood pressure. However, whether ATRAP is differentially expressed in Spontaneously Hypertensive Rats (SHR) is unknown. We hypothesized that ATRAP gene and protein expression decreases during the development of hypertension in SHRs, leading to increased Ang II signaling. To test this, we used male SHRs at 4 (pre-hypertensive) and 12-weeks-old (wo, hypertensive; mean arterial pressure (MAP) 128.0±2.7 vs. 149.2±5.30 mmHg, respectively, measured by tail-cuff recording [Kent Scientific]) and age-matched normotensive Wistar rats (WIS, 92.6±3.3 vs. 123.6±4.6 mmHg, respectively). We measured ATRAP and AT1R mRNA expression in PVN samples using real-time PCR. Protein expression density of ATRAP was assessed using immunofluorescence (IF) and confocal imaging. We found no differences in ATRAP mRNA expression in the PVN between groups ( p >0.05). On the other hand, we found a significant increase in AT1R mRNA expression after hypertension is developed (WIS 12wo: 1.12 ± 0.35; vs. SHR 12wo: 4.17 ± 1.07, p =0.02). While no differences were found in ATRAP, using ATRAP/AT1R ratio as an indicator of AT1R overactivity and decrease in ratio after the development of hypertension (SHR 12wo: 0.23 ± 0.06 vs. WIS 12wo:1.05 ± 0.25) further indicate increased AngII signaling. As observed with ATRAP mRNA expression, no statistical differences were observed in ATRAP protein density within the SHR at 12wo as measured in a separate cohort of animals (MAP SHR 12wo:149.0 ± 3.7 vs. WIS 12wo:115.8 ± 4.0; p >0.05). These results suggest that the increased AngII signaling within the PVN of SHR are most likely due to increased AT1r expression rather than a differential expression of ATRAP. This work was partially funded by AHA 953524 to VCB.

GLP1 receptor agonists exhibit antioxidant and anti-inflammatory properties in human heart tissue via the canonical GLP1 receptor expression through a AMPK-mediated pathway

Abstract Background Clinical research has demonstrated that glucagon-like peptide-1 receptor (GLP1R) agonists (GLP1RA) can decrease the occurrence of major cardiovascular events in diabetic patients, regardless of their glycemic control. However, a significant challenge in understanding the positive effects of GLP1RA has been the lack of knowledge regarding the specific cellular types that express GLP1R. Despite the established cardiovascular benefits of GLP1RA, previous studies have been unable to confirm the expression of GLP1R in the heart. Aim The study aimed to investigate the expression and function of GLP1R in heart tissue. Methods We collected human cardiac tissue samples from 30 patients who underwent left ventricle (LV) biopsies (n=21) or right atrial (RA) appendage closure (n=9) at a university hospital. Gene expression levels were assessed using RT-qPCR, protein levels by Western blot analysis, in situ tissue localization of proteins by immunofluorescence (IF) staining, and the level of oxidative stress using dihydroethidium. Results LV biopsies and RA appendages showed a substantial heterogeneity in GLP1R mRNA levels. GLP1R mRNA levels exhibited a positive correlation with IL1B, IL6, TNFα, CCL2, CD68, ACE1, AT1R, and VCAM1 mRNA levels, but a negative correlation with NOS3 mRNA levels. High GLP1R expression in both RA appendages and LV biopsies was associated with increased oxidative stress levels, which were mitigated by the antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor (VAS-2870), ACE inhibitor (perindoprilat), AT1R antagonist (losartan), TNF-α receptor neutralizing antibody (infliximab) and by GLP1R agonists (liraglutide and semaglutide) with inhibitory effects amounting to about 50-60 %. Exendin, a GLP1R antagonist reversed the effect of liraglutide and semaglutide which accounted for a receptor-mediated antioxidant effect. H-89, a PKA inhibitor equally reversed liraglutide and semaglutide effect on ROS generation suggesting a canonical GLP1R mediated effect involving the AMPK pathway. Conclusion These findings indicate that human cardiac GLP1R expression levels are correlated to low-grade inflammation and endothelial dysfunction. Increased expression levels of GLP1R were associated with higher levels of oxidative stress sensitive to GLP1RA in a canonical GLP1R AMPK-mediated manner. Therefore, the cardiovascular beneficial effects of GLP1RA might be attributable to their antioxidant and anti-inflammatory properties, specifically within the cardiac system.

Abstract 005: Renal Macrophages From Losartan Treated Mice Transfer The Hypertension

The immune system participates in the Angiotensin II (AngII)-dependent hypertension (HTN). Dendritic Cells (DCs)-CD11c + and macrophages (Mo) are abundant in the kidney. However, new subpopulations of CD11c + cells with Mo characteristics (F4/80 + ) have been described. Recently, studies have shown that type-1 AngII receptor (AT 1 R) in CD11c + cells could have a protective effect on HTN in contrast to the pro-hypertensive effects of AT 1 R activation in other cells. Here, our goal was to evaluate whether renal Mo (rMo) modify their phenotype during AngII-induced inflammatory kidney damage, and whether their transfer to normotensive animals promotes HTN in an AT 1 R-dependent manner. Male C57BI/6 mice (10 weeks, n=6) were infused with AngII (490 ng/kg/min), AngII+Losartan (LOS; 20mg/kg/d, in drinking water), or vehicle (Vh) for 14-days. Systolic blood pressure (SBP) was measured by plethysmography. Renal DCs (rDCs, CD11c + F4/80 - ) and rMo (CD11c + F4/80 + ) were evaluated by flow cytometry and isolated at 14-days for the transfer to normotensive mice, which were followed for 7-days (n=4-5). Finally, rDCs and rMo were stimulated in vitro with AngII (100nM, 24h) to analyze the mRNAs abundance of AT 1 R. All data are presented by mean±standard deviation.LOS prevented the SBP rise (AngII+LOS=110.3±11.1 vs. AngII=134.7±6.9 mmHg; p<0.01) and the increase of rDCs population (AngII+LOS=12.8±2.0% vs. AngII=16.5±0.6%; p<0.01) induced by AngII, without changing rMo population. The rMo transfer from AngII mice did not induce HTN in normotensive animals after 24h (basal=106.8±2.3 vs. 112.4±4.9 mmHg; ns), compared to rDCs (Basal=110.0±8.3 vs. 132.0±14.3 mmHg; p<0.05). However, the rMo from AngII+LOS mice increased SBP in healthy animals compared to their baseline (basal=109±5.6 vs. 123±8.0 mmHg; p<0.05). This effect was not observed by rDCs transfer (basal=109±2.9 vs. 113±7.0 mmHg; ns). Finally, the rMo from AngII mice showed an increase in the AT 1 R mRNA abundance (p<0.05), compared to Vh rMo, however, AngII in vitro treatment did not modify the mRNA levels.The in vivo AT 1 R antagonism may have a differential effect on rMo, transferring HTN to normotensive mice. These findings suggest that subpopulations of renal CD11c + cells have a differential role in HTN.Fondecyt 1231909

G-protein-coupled receptor MAS deletion produces a preeclampsia-like phenotype in FVB/N mice.

An unbalance in the renin-angiotensin (Ang) system (RAS) between the Ang II/AT1 and Ang-(1-7)/Mas axis appears to be involved in preeclampsia (PE), in which a reduction in Ang-(1-7) was observed. Here, we tested whether the reduction in the activity of the Ang-(1-7)/Mas axis could be a contributing factor for the development of PE, using Mas-deficient (Mas-/-) mice. Cardiovascular parameters were evaluated by telemetry before, during pregnancy and 4 days postpartum in 20-week-old Mas-/- and wild-type (WT) female mice. Mas-/- mice presented reduced arterial blood pressure (BP) at baseline (91.3 ± 0.8 in Mas-/- vs. 94.0 ± 0.9 mmHg in WT, Diastolic, P<0.05). However, after the 13th day of gestation, BP in Mas-/- mice started to increase, time-dependently, and at day 19 of pregnancy, these animals presented a higher BP in comparison with WT group (90.5 ± 0.7 in Mas-/- vs. 80.3 ± 3.5 mmHg in WT, Diastolic D19, P<0.0001). Moreover, pregnant Mas-/- mice presented fetal growth restriction, increase in urinary protein excretion as compared with nonpregnant Mas-/-, oliguria, increase in cytokines, endothelial dysfunction and reduced ACE, AT1R, ACE2, ET-1A, and eNOS placental mRNA, similar to some of the clinical manifestations found in the development of PE. These results show that Mas-deletion produces a PE-like state in FVB/N mice.

Immunolocalization of the AT-1R Ang II Receptor in Human Kidney Cancer.

This study aimed to evaluate AT1-R expression in normal and cancerous human kidneys, how these expressions are modified, and AT1-R functionality. AT-1R mRNA expression, determined by real-time PCR, was detected in all samples. AT-1R mRNA increased in well-differentiated cancer (G1, p < 0.01) and decreased 2.9-fold in undifferentiated cancer (G4, p < 0.001) compared with normal kidney tissues. Immunocytochemistry analysis showed that the AT-1R was expressed in the normal tubular epithelium. The glomerulus was also immunoreactive, and as expected, the smooth muscle cells of the vessel walls also expressed the receptor. A total of 35 out of 42 tumors were AT-1R positive, with the cell tumors showing varying numbers of immunoreactive cells, which were stained in a diffuse cytoplasmic and membranous pattern. Computer-assisted counting of the stained tumor cells showed that the number of AT-1R-positive cells increased in the well-differentiated cancers. The functionality of AT-1R was assessed in primary cultures of kidney epithelial cells obtained from three G3 kidney cancer tissues and corresponding histologically proven non-malignant tissue adjacent to the tumor. Indeed, Ang II stimulated, in a dose-dependent manner, the 24 h proliferation of normal kidney cells and cancer cells in the primary culture and phosphorylated extracellular regulated kinases 1 and 2. In conclusion, Ang II may be involved in the growth or function of neoplastic kidney tissue.

Up-regulation of the kidney renin angiotensin system contributes to long-term renal dysfunction in severe food restricted-refed female rats

Background: Severe food restriction (sFR) due to various psychological, environmental, and economical reasons can have adverse consequences to cardiovascular functioning and health. Less understood are the long-term risks for developing cardiovascular disease after the sFR period has ended. The renin-angiotensin system (RAS) is responsible for regulating blood pressure and we have found that the RAS is chronically upregulated months after the sFR is over and body weight (BW) recovered due to refeeding (sFR-Refed). AIM: Determine the role of the kidney in the chronic up-regulation of the RAS in sFR-Refed rats. Methods: We investigated the long-term consequences of sFR after refeeding on kidney structure and function in sFR-Refed rats. Female Fischer rats (3-months-old) were maintained on normal chow (Ctrl) ad libitum or a 60% caloric restricted diet for 2 weeks. Thereafter, all rats received regular chow ad libitum for 3 months. Kidney function was analyzed by precision ultrasound. ACE expression (qPCR) and activity (fluorescent assay) were measured and renal pathology was assessed by H&amp;E staining. Results: After 2 weeks of sFR, rats lost 15% of their initial BW [DFinal/Initial): Ctrl, 1.50±0.80 vs sFR, -15.4±1.1; p&lt;0.001; n=8]. After 3 months of refeeding, there was no detectable difference in BW, blood pressure and heart rate between Ctrl and sFR-Refed groups. However, the renal artery blood flow was reduced by 13% [(mm/s): Ctrl, 255 ± 12 vs sFR-Refed, 199 ± 7.8; p&lt;0.02; n=4-8 Glomeruli size was reduced [(mm): Ctrl, 399 ± 6.2 vs sFR-Refed, 383 ± 3.8; p&lt;0.05; n=9] and renal AT1R mRNA expression was increased by 1.3-fold [(fold of Ctrl): Ctrl, 1.00 ± 0.060 vs sFR-Refed, 1.29 ± 0.040; p&lt;0.005; n=8]. Conclusion: In summary, AT1Rs in the renal cortex are up-regulated under conditions in which kidney structure and function are impaired months after the sFR period has ended and BW is restored to normal levels. These findings suggest increased renal AT1R activity contributes to the long-term renal dysfunction observed in sFR-Refed rats. Further research is needed to understand how women who are subjected either voluntarily (e.g., crash diets) or involuntarily (e.g., very low food security) to periods of inadequate caloric intake could be at increased risk for developing renal disease later in life. AHA: 940246 (AS); IH 1R01HL119380 (KS) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PS-B12-10: CLASSIC CARDIOVASCULAR PROTECTIVE SIGNALING IN ANGIOTENSIN II-INDUCED NOVEL PREECLAMPSIA RAT MODEL

Angiotensin II (Ang II)-induces vasoconstriction, in vivo perfusion has been shown to induce preeclampsia (PE) in pregnant rats. In this study, we aimed to examine the protein and mRNA expressions of endothelial and neuronal NOS (eNOS, nNOS) as well as angiotensin II type 2 receptor (AT2R) in normal pregnant and Ang II-induced PE rats. Pregnant (8 weeks) and non-pregnant SD rats (8 weeks, GD-8) were infused with Ang II via an osmotic minipump (1 ug/kg/min), and the rats (GD-21) were sacrificed 13 days later. Pregnant and non-pregnant rats without Ang II were taken as sham controls. The fetal weight, crown-rump length and placental weight of PE rats were significantly lower compared to those without Ang II infusion. Immunohistochemistry results showed an approximately 30% increase in glomerular volume, suggesting morphological changes in the kidney and Ang II infusion exhibits a PE phenotype. Ang II infusion did not affect AT2R, AT1R and nNOS protein or mRNA expression in the uterus of non-pregnant or pregnant rats. However, eNOS mRNA expression was reduced in PE rats (p &lt; 0.0001) although protein expression was not affected by Ang II infusion. However, in the placenta, AT1R and AT2R protein expression were not different in PE, but AT2R mRNA was decreased (p &lt; 0.0001). eNOS protein was only expressed in fetal vascular endothelial cells, and eNOS protein expression was higher in PE rats (P = 0.001) but eNOS mRNA was decreased (p = 0.0096). Notably, strong nNOS expression was observed in placental trophoblast cell groups of sham and PE rats, and both mRNA and protein expression of nNOS was decreased in placental lysates of Ang II rats (respectively p = 0.0287 and p &lt; 0.0001). These results demonstrate that we have recapitulated the PE model of Ang II infusion in rats. The expression of nNOS was reduced, and the localization of nNOS in the trophoblast suggested a new functional remodeling in the fetal vasculature. In the placenta, decreased expression of AT2R and nNOS may be a novel mechanism of impaired fetal development.

CLASSIC CARDIOVASCULAR PROTECTIVE SIGNALING IN ANGIOTENSIN II-INDUCED NOVEL PREECLAMPSIA RAT MODEL

Objective: Angiotensin II (Ang II)-induces vasoconstriction, in vivo perfusion has been shown to induce preeclampsia (PE) in pregnant rats. In this study, we aimed to examine the protein and mRNA expressions of endothelial and neuronal NOS (eNOS, nNOS) as well as angiotensin II type 2 receptor (AT2R) in normal pregnant and Ang II-induced PE rats. Design and method: Pregnant (8 weeks) and non-pregnant SD rats (8 weeks, GD-8) were infused with Ang II via an osmotic minipump (1 ug/kg/min), and the rats (GD-21) were sacrificed 13 days later. Pregnant and non-pregnant rats without Ang II were taken as sham controls. The fetal weight, crown-rump length and placental weight of PE rats were significantly lower compared to those without Ang II infusion. Immunohistochemistry results showed an approximately 30% increase in glomerular volume, suggesting morphological changes in the kidney and Ang II infusion exhibits a PE phenotype. Results: Ang II infusion did not affect AT2R, AT1R and nNOS protein or mRNA expression in the uterus of non-pregnant or pregnant rats. However, eNOS mRNA expression was reduced in PE rats (p &lt; 0.0001) although protein expression was not affected by Ang II infusion. However, in the placenta, AT1R and AT2R protein expression were not different in PE, but AT2R mRNA was decreased (p &lt; 0.0001). eNOS protein was only expressed in fetal vascular endothelial cells, and eNOS protein expression was higher in PE rats (P = 0.001) but eNOS mRNA was decreased (p = 0.0096). Notably, strong nNOS expression was observed in placental trophoblast cell groups of sham and PE rats, and both mRNA and protein expression of nNOS was decreased in placental lysates of Ang II rats (respectively p = 0.0287 and p &lt; 0.0001). Conclusions: These results demonstrate that we have recapitulated the PE model of Ang II infusion in rats. The expression of nNOS was reduced, and the localization of nNOS in the trophoblast suggested a new functional remodeling in the fetal vasculature. In the placenta, decreased expression of AT2R and nNOS may be a novel mechanism of impaired fetal development.

Endothelial dysfunction after androgen deprivation therapy and the possible underlying mechanisms.

Androgen deprivation therapy (ADT) is a key treatment modality in the management of prostate cancer (PCa), especially for patients with metastatic disease. Increasing evidences suggest that patients who received ADT have increased incidence of diabetes, myocardial infarction, stroke, and even mortality. It is important to understand the pathophysiological mechanisms on how ADT increases cardiovascular risk and induces cardiovascular events, which would provide important information for potential implementation of preventive measures. Twenty-six 12-week-old male SD rats were divided into four groups for different types of ADTs including: the bilateral orchidectomy group (Orx), LHRH agonist group (leuprolide), LHRH antagonist group (degarelix), and control group. After treated with drug or adjuvant injection every 3 weeks for 24 weeks, all rats were sacrificed and total blood were collected. Aorta, renal arteries, and kidney were preserved for functional assay, immunohistochemistry, western blot, and quantitative reverse-transcription polymerase chain reaction. In vascular reactivity assays, aorta, intrarenal, and coronary arteries of all three ADT groups showed endothelial dysfunction. AT1R and related molecules at protein and messenger RNA (mRNA) level were tested, and AT1R pathway was shown to be activated and played a role in endothelial dysfunction. Both ACE and AT1R mRNA levels were doubled in the aorta in the leuprolide group while Orx and degarelix groups showed upregulation of AT1R in the kidney tissues. By immunohistochemistry, our result showed higher expression of AT1R in the intrarenal arteries of leuprolide and degarelix groups. The role of reactive oxygen species in endothelial dysfunction was confirmed by DHE fluorescence, nitrotyrosine overexpression, and upregulation of NOX2 in the different ADT treatment groups. ADT causes endothelial dysfunction in male rats. GnRH receptor agonist compared to GnRH receptor antagonist, showed more impairment of endothelial function in the aorta and intrarenal arteries. Such change might be associated with upregulation and activation of AngII-AT1R-NOX2 induced oxidative stress in the vasculature. These results help to explain the different cardiovascular risks and outcomes related to different modalities of ADT treatment.

고강도 인터벌 운동이 난소절제 쥐의 혈중 지질과 레닌-안지오텐신 시스템(RAS) 관련 인자에 미치는 영향

In this study, we investigated the effects of high-intensity interval exercise on blood lipids and renin-angiotensin system (RAS)-related factors in ovariectomized rats. The rats were categorized into a control group (CON, n=10), an ovariectomized group (OVA, n=10), and an ovariectomized+high-intensity exercise group (OVA+HIE, n=10). High-intensity interval exercise was performed on a treadmill for 30 min per session, once a day for 12 weeks. Data was analysed using one-way analysis of variance (ANOVA). Compared with the other groups, the total cholesterol, triglyceride, and low-density lipoprotein cholesterol levels were significantly reduced and the high-density lipoprotein cholesterol levels were significantly increased in the OVA+TE group after 12 weeks of high-intensity interval exercise. Additionally, ACE1 and AT1R mRNA expression were significantly reduced in the OVA+TE group. However, no significant intergroup difference was observed in SERCA2a mRNA expression. These results highlight that 12-week high-intensity interval exercise produced a positive effect on blood lipids and RAS-related factors in ovariectomized rats.

Effect of Fosfomycin on Cyclosporine Nephrotoxicity.

Fosfomycin (Fos) has emerged as a potential treatment against multidrug-resistant organisms, however, there has been little work done on its influence on calcineurin inhibitor nephrotoxicity (CIN). This study was designed to evaluate the effect of Fos in combination with cyclosporine (CsA) on CIN. Two sets of experiments were undertaken. In the first, Wistar rats received different doses of Fos: 0, 62.5, 125, 250, and 500 mg/kg. In the second, rats were divided into four groups: control, CsA 15 mg/kg s.c., CsA + fosfomycin 62.5 mg/kg (CsA + LF), and CsA + Fos 500 mg/kg (CsA + HF). CsA was administrated daily for 14 days, whereas Fos administration started on the ninth day followed by two more doses, delivered 48 h apart. The administration of different Fos doses did not alter renal function. In contrast, CsA induced arteriolopathy, hypoperfusion, a reduction in the glomerular filtration rate, and downregulation of eNOS, angiotensinogen, and AT1R mRNA levels. Lower doses of Fos did not modify CIN. Instead, the CsA + HF group exhibited greater hypoperfusion, arteriolopathy, and oxidative stress, and increased mRNA levels of pro-inflammatory cytokines. This study shows that Fos administered by itself at different doses did not cause renal injury, but when it was given repeatedly at high dosages (500 mg/kg) in combination with CsA, it increased CIN through the promotion of greater oxidative stress and renal inflammation.

Hypogonadal hypertension in male Sprague-Dawley rats is renin-angiotensin system-dependent: role of endogenous androgens

BackgroundAcutely, testosterone (TES) and other androgens are efficacious vasodilators, both in vitro and in vivo; however, their long-term effects on arterial blood pressure (BP) remain unclear. It was hypothesized that endogenous androgens exert long-term anti-hypertensive effects on systemic BP through a combination of genomic and nongenomic effects to enhance vasodilation of the systemic vasculature.MethodsThe long-term effects of endogenous TES and exogenous TES replacement therapy (TRT) on BP were studied in intact (InT) and castrated (CsX) male Sprague-Dawley (SD) and testicular-feminized male (Tfm, androgen receptor defective) rats (12 weeks old). Systolic BP (tail-cuff plethysmography) was determined weekly for 15 weeks in InT-control and CsX rats. Some CsX-SD rats received androgen replacement therapy at 10-15 weeks with TES-enanthate (TRT; 1.75 mg/kg, 2x/week) or DHT-enanthate (DRT; 1.00 mg/kg. 2x/week) and a separate group of CsX-SD rats received losartan-potassium in drinking water (LST, 250 mg/L) for the entire 15 week period. Expression of renin, angiotensinogen (Agt), angiotensin converting enzyme (ACE), and angiotensin II type I receptor (AT1R) mRNA in kidney and aorta were determined by real-time PCR (rt-PCR) and plasma renin levels were determined by radioimmunoassay.ResultsThere was a progressive rise in BP over 10 weeks in CsX (109 ± 3.3 vs. 143 ± 3.5 mmHg), while BP remained stable in InT-control (109 ± 3.0 vs. 113 ± 0.3). BP gradually declined to normal in CsX-TRT rats (113 ± 1.3), while BP remained elevated in CsX (140 ± 1.2) and normal in InT-control (113 ± 0.3). LST prevented the development of hypertension in CsX at 10 weeks (100 ± 1.5 in CsX + LST vs. 143 ± 3.5 in CsX). During the next 5 weeks with TES-RT, BP declined in CsX-TRT (113 ± 1.3) and remained lower in CsX + LST (99 ± 0.4). DHT-RT reduced BP in CxS to a similar extent. In Tfm, CsX resulted in a similar rise in BP (109 ± 0.7 vs. 139 ± 0.4 mmHg), but TRT reduced BP more rapidly and to a greater extent (106 ± 2.8). rt-PCR of the kidney revealed that CsX increased expression of mRNA for renin (92%), ACE (58%), and AT1R (80%) compared to InT, while TES RT normalized expression of renin, AT1R, and ACE mRNA to levels of InT rats. Plasma renin levels exhibited changes similar to those observed for renin mRNA expression.ConclusionsThis is the first study to examine the long-term effects of endogenous and exogenous androgens on BP in male SD and Tfm rats. These data reveal that endogenous androgens (TES) exert anti-hypertensive effects that appear to involve non-genomic and possibly genomic mechanism(s), resulting in reductions in RAS expression in the kidney and enhanced systemic vasodilation.

Anabolic steroid excess promotes hydroelectrolytic and autonomic imbalance in adult male rats: Is it enough to alter blood pressure?

AimThe present study investigated the effects of anabolic steroid (AS) excess on blood pressure regulation. MethodsMale Wistar rats were treated with nandrolone decanoate (AS) or vehicle (CTL) for 8 or 10 weeks. Saline (1.8%) and water intake were measured in metabolic cages. Urinary volume, osmolarity, Na+ and K+ concentrations, and plasma osmolarity were measured. The autonomic balance was estimated by heart rate variability at baseline or after icv injection of losartan. Cardiac function was assessed by echocardiography and ex vivo recordings. Myocardial collagen deposition was evaluated by Picrosirius-Red staining. Vascular reactivity and wall thickness were investigated in aortic sections. Blood pressure (BP) was assessed by tail-cuff plethysmography. Angiotensin II type I receptor (AT1R), renin, and mineralocorticoid receptor (MR) mRNA expression was measured in the kidneys and whole hypothalamus. ResultsAS group exhibited decreased urinary volume and Na+ concentration, while urinary K+ concentration, plasma osmolarity, and renal AT1R and renin mRNA levels were increased compared to CTL (p < 0.05). Water intake was increased, and saline intake was decreased in the AS group (p < 0.01). AS group exhibited increased low-frequency/high-frequency-ratio, while it was decreased by icv injection of losartan (p < 0.05) compared to baseline. Neither cardiac function nor vascular reactivity/morphology was affected by AS excess (p > 0.05). Ultimately, BP levels were not altered by AS excess (p > 0.05). ConclusionAS excess promoted hydroelectrolytic and autonomic imbalance but did not alter vascular or cardiac function/morphology.

Expressions of Renin, angiotensin converting enzyme, angiotensin receptor 1, and angiotensin receptor 2 in synovial tissue of osteoarthritis at different stages

To study the expressions of Renin, angiotensin converting enzyme (ACE), angiotensin receptor 1 (AT1R), and AT2R in synovial tissue of osteoarthritis (OA) at different stages. The patients who were treated with upper knee amputation because of trauma or total knee arthroplasty for OA between January 2018 and December 2018 were enrolled. Among them, 32 patients who met the selection criteria were included in the study. According to the Kellgren-Lawrence (K-L) X-ray classification, they were allocated to normal synovial group (group A, n=9), moderate OA synovial group (group B, n=11, K-L level 3), and advanced OA synovial group (group C, n=12, K-L level 4). The relative expressions of Renin, ACE, AT1R, and AT2R mRNAs and proteins were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot. The relative expressions of Renin, ACE, and AT1R mRNAs and proteins were significantly higher in group B and group C than in group A ( P<0.05). The relative expressions of ACE and AT1R mRNAs and proteins and Renin protein were significantly higher in group C than in group B ( P<0.05). However, the relative expressions of AT2R mRNA and protein were lower in group B and group C than in group A ( P<0.05), and in group C than in group B ( P<0.05). The expressions of Renin, ACE, and AT1R in synovial tissue of osteoarthritis significantly increase as the K-L level increased, and the expression of AT2R decreases. Renin, ACE, AT1R, and AT2R have a certain degree of correlation with the development of OA.

Ang-(1-7) treatment attenuates lipopolysaccharide-induced early pulmonary fibrosis

Early pulmonary fibrosis is the leading cause of poor prognosis in patients with acute respiratory distress syndrome (ARDS). However, whether the renin-angiotensin system (RAS) can serve as a therapeutic target is unknown. In this study, an animal model of early pulmonary fibrosis was established via the LPS three-hit regimen. Afterwards, the animals were treated with intraperitoneal injections of Ang-(1-7), AVE0991, or A779 once per day for 20 days. The plasma and BALF AngII levels of the animals were increased, while there were no significant changes in Ang-(1-7) levels in lung tissue after LPS treatment. Furthermore, the AT1R protein levels were significantly increased and the Mas levels were significantly decreased on days 14 and 21. Administration of Ang-(1-7) downregulated LPS-induced AT1R mRNA expression, which was upregulated by A779. The expression of Mas mRNA responded in the opposite direction relative to AT1R. Moreover, LPS caused decreased levels of Mas and E-cadherin and increased AT1R, Vimentin, and Src phosphorylation levels. Ang-(1-7) or AVE0991 blocked these effects but was counteracted by A779 treatment. Our findings suggested that AngII and AT1R levels exhibit opposite dynamic trends during LPS-induced early pulmonary fibrosis, as do Ang-(1-7) and Mas. Ang-(1-7) exerts protective effects against early pulmonary fibrosis, mainly by regulating the balance between AngII and AT1R and between Ang-(1-7) and Mas and by inhibiting Src kinase activation.

Differential Effects of the Mitochondria-Active Tetrapeptide SS-31 (D-Arg-dimethylTyr-Lys-Phe-NH2) and Its Peptidase-Targeted Prodrugs in Experimental Acute Kidney Injury.

The mitochondria-active tetrapeptide SS-31 can control oxidative tissue damage in kidney diseases. To investigate other potential beneficial nephroprotective effects of SS-31, in vivo murine models of acute tubular injury and glomerular damage were developed. Reduction of acute kidney injury was demonstrated in mice treated with SS-31. The expression of mRNAs involved in acute inflammatory and oxidative stress responses in the diseased kidneys confirmed that SS-31 could regulate these pathways in our in vivo models. Furthermore, ex vivo histoenzymography of mouse kidneys showed that aminopeptidase A (APA), the enzyme involved in the processing of angiotensin (Ang) II to Ang III, was induced in the diseased kidneys, and its activity was inhibited by SS-31. As the renin–angiotensin system (RAS) is a main regulator of kidney functions, the modulation of Ang receptors (ATR) and APA by SS-31 was further investigated using mRNAs extracted from diseased kidneys. Following acute tubular and/or glomerular damage, the expression of the AT1R mRNA was upregulated, which could be selectively downregulated upon SS-31 administration to the animals. At the same time, SS-31 was able to increase the expression of the AT2R, which may contribute to limit renal damage. Consequently, SS-31-based prodrugs were developed as substrates and/or inhibitors for APA and were screened using cells expressing high levels of APA, showing its selective regulation by α-Glu-SS-31. Thus, a link between SS-31 and the RAS opens new therapeutic implications for SS-31 in kidney diseases.

Cutaneous inflammation differentially regulates the expression and function of Angiotensin-II types 1 and 2 receptors in rat primary sensory neurons.

Neuropathic and inflammatory pain results from cellular and molecular changes in dorsal root ganglion (DRG) neurons. The type-2 receptor for Angiotensin-II (AT2R) has been involved in this type of pain. However, the underlying mechanisms are poorly understood, including the role of the type-1 receptor for Angiotensin-II (AT1R). Here, we used a combination of immunohistochemistry and immunocytochemistry, RT-PCR and in vitro and in vivo pharmacological manipulation to examine how cutaneous inflammation affected the expression of AT1R and AT2R in subpopulations of rat DRG neurons and studied their impact on inflammation-induced neuritogenesis. We demonstrated that AT2R-neurons express C- or A-neuron markers, primarily IB4, trkA, and substance-P. AT1R expression was highest in small neurons and co-localized significantly with AT2R. In vitro, an inflammatory soup caused significant elevation of AT2R mRNA, whereas AT1R mRNA levels remained unchanged. In vivo, we found a unique pattern of change in the expression of AT1R and AT2R after cutaneous inflammation. AT2R increased in small neurons at 1day and in medium size neurons at 4days. Interestingly, cutaneous inflammation increased AT1R levels only in large neurons at 4days. We found that in vitro and in vivo AT1R and AT2R acted co-operatively to regulate DRG neurite outgrowth. In vivo, AT2R inhibition impacted more on non-peptidergic C-neurons neuritogenesis, whereas AT1R blockade affected primarily peptidergic nerve terminals. Thus, cutaneous-induced inflammation regulated AT1R and AT2R expression and function in different DRG neuronal subpopulations at different times. These findings must be considered when targeting AT1R and AT2R to treat chronic inflammatory pain. Cover Image for this issue: doi: 10.1111/jnc.14737.

Hypogonadal Hypertension in Male Sprague‐Dawley Rats Is Reversed by Testosterone Replacement Therapy, which Suppresses Renin‐Angiotensin System Expression

Testosterone (TES) and other androgens are efficacious vasodilators acutely, both in vitro and in vivo; however, their long‐term effects on arterial blood pressure (BP) are unclear. We previously reported that castration (CsX) produced hypertension in normal male rats, and that TES replacement therapy (TRT) was antihypertensive and normalized BP. Thus, long‐term effects of endogenous TES and exogenous TRT on BP and renin‐angiotensin system (RAS) function were studied in intact (InT) and castrated (CsX) male Sprague‐Dawley (SD) and Testicular‐feminized male (Tfm, androgen receptor defective) rats (12–13 wk old). Weekly measurements of systolic BP (tail cuff plethysmography) revealed a progressive rise in BP over 10 wks in CsX (108 ± 0.9 vs. 139 ± 1.2 mmHg), while BP remained stable in InT (109 ± 3.1 vs. 115 ± 0.5). During the next 5 weeks, half of CsX received TRT (CsX+TES‐enanthate‐replaced; 1.75 mg/kg, 2X/wk). BP gradually declined to normal in CsX+TES replaced rats (115 ± 1.2), while BP remained elevated in CsX (141 ± 1.2) and normal in InT (113 ± 0.3). In separate CsX‐SD rats, treatment with Losartan (LST; 250 mg/L drinking water) prevented development of hypertension at 10 wks (95 ± 0.8 CsX+LST vs. 139 ± 1.2 in CsX). During the next 5 weeks with TRT, BP declined in CsX+TES (113 ± 1.3) and remained lower in CsX+LST (99 ± 0.4). In Tfm, CsX resulted in a similar rise in BP (108 ± 0.6 vs. 139 ± 0.4 mmHg), and TRT reduced BP to a similar extent (106 ± 1.5). Real‐time PCR (rt‐PCR) of kidney from InT, CsX, and CsX+TES rats revealed that CsX increased expression of renin (45.5%), AT1 receptor (AT1R; 38%), and angiotensin converting enzyme (ACE; 239%) mRNA, while TRT normalized expression of renin, AT1R, and ACE mRNA to levels of InT rats. In contrast, CsX reduced expression of angiotensinogen (Angt) mRNA (59%) while TRT restored Angt mRNA to 78% of InT rats. 24 hr urine output (UO) in InT‐SD was 48.3 ml/kg at baseline, 34.3 at 10 wks, 38.4 at 12 wks, and 34.8 at 15 wks. UO in CsX‐SD was 47.6, 30.3, 25.2, and 27.7; UO in CsX+TRT was 46.1, 29.2 36.2, and 29.5. These data suggest that: 1) endogenous androgens (TES) exert antihypertensive effects in male SD and Tfm rats; 2) the antihypertensive effect of TES appear to involve a diuretic effect on the kidney, which is non‐genomically mediated; and 3) these antihypertensive effects may involve TES‐induced reductions in RAS expression in the kidney.Support or Funding InformationState of TexasThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetic disruption of guanylyl cyclase/natriuretic peptide receptor-A upregulates renal (pro) renin receptor expression in Npr1 null mutant mice

The objective of the present study was to determine whether targeted-disruption of Npr1 gene (encoding for guanylyl cyclase/natriuretic peptide receptor-A; GC-A/NPRA) upregulates pro(renin) receptor (P)RR expression and leads to the activation of MAPKs in Npr1 gene-knockout mice. The Npr1 homozygous (Npr1−/−; 0-copy), heterozygous (Npr1+/−; 1-copy), wild-type (Npr1+/+; 2-copy), and gene-duplicated (Npr1++/++; 4-copy) mice were utilized. To identify the canonical pathway of (P)RR, we administered ACE-1 inhibitor (captopril), AT1R blocker (losartan), and MAPKs inhibitors (U0126 and SB203580) to all Npr1 mice genotypes. The renal expression of (P)RR mRNA was increased by 3-fold in 0-copy mice and 2-fold in 1-copy mice compared with 2-copy mice, which was also associated with significantly increased expression of ACE-1 and AT1R mRNA levels. Similarly, the phosphorylation of MAPKs (Erk1/2 and p-p38) was enhanced by 3.5-fold and 3.2-fold, respectively, in 0-copy mice with significant increases in 1-copy mice compared with 2-copy mice. The kidney and plasma levels of proinflammatory cytokines were significantly elevated in 0-copy and 1-copy mice. Treatment with captopril and losartan did not alter the expression of (P)RR in any of the Npr1 mice genotypes. Interestingly, losartan significantly reduced the phosphorylation of Erk1/2 and p38 in Npr1 mice. The present results suggest that the ablation of Npr1 upregulates (P)RR, MAPKs (Erk1/2 and p38), and proinflammatory cytokines in 0-copy and 1-copy mice. In contrast, the duplication of Npr1 exhibits the anti-inflammatory and antihypertensive effects by reducing the activation of MAPKs and inhibiting the expression levels of RAAS components and proinflammatory cytokines.