Angiotensin AT1 Research Articles

Therapeutic opportunities in targeting the protective arm of the renin-angiotensin system to improve insulin sensitivity: a mechanistic review.

In recent years, the knowledge of the physiological and pathophysiological roles of the renin-angiotensin system (RAS) in glucose metabolism has advanced significantly. It is now well-established that blockade of the angiotensin AT1 receptor (AT1R) improves insulin sensitivity. Activation of the AT2 receptor (AT2R) and the MAS receptor are significant contributors to this beneficial effect. Elevated availability of angiotensin (Ang) II) for interaction with the AT2R and increased Ang-(1-7) formation during AT1R blockade mediate these effects. The ongoing development of selective AT2R agonists, such as compound 21 and the novel Ang III peptidomimetics, has significantly advanced the exploration of the role of AT2R in metabolism and its potential as a therapeutic target. These agents show promise, particularly when RAS inhibition is contraindicated. Additionally, other RAS peptides, including Ang IV, des-Asp-Ang I, Ang-(1-9), and alamandine, hold therapeutic capability for addressing metabolic disturbances linked to type 2 diabetes. The possibility of AT2R heteromerization with either AT1R or MAS receptor offers an exciting area for future research, particularly concerning therapeutic strategies to improve glycemic control. This review focuses on therapeutic opportunities to improve insulin sensitivity, taking advantage of the protective arm of the RAS.

Abstract 66: Early-Life Sodium Restriction Programs Salt-Sensitivity of Autonomics and Blood Pressure in Adult C57BL/6J Mice

Extremely premature birth is a risk factor for sodium (Na) depletion due to renal immaturity and urine Na losses. We have proposed that Na depletion in preterm infants mechanistically contributes to their increased risk for cardiovascular and metabolic disease observed in adulthood, potentially due to changes during hypothalamic development. In humans, the hypothalamus matures between 20-25 weeks of gestation; in mice, this developmental window occurs between postnatal day (PD) 21 and PD42. Previously, we demonstrated in mice that dietary Na restriction from PD21 to PD42 induced long-lasting increases in energy expenditure via exaggerated autonomic activity. Therefore, we hypothesized that restricting dietary Na from PD21 to PD42 would similarly induce programmed changes in cardiovascular control via changes in autonomic and neurohypophyseal functions. Male C57BL/6J mice were provided a customized low (0.04%, n=7) or supplemented (0.30%, n=8) Na version of the soy protein-free Teklad 2920x diet from PD21 to PD42, after which they were returned to the standard (0.15% Na) 2920x diet. Radiotelemetric blood pressure (BP) transducers were implanted at 7-8 weeks of age. At 10 weeks of age, no differences in BP or heart rate (HR) were noted between groups. All animals were switched to a high (1%) Na diet at 12 weeks of age. At 16 weeks of age, systolic BP was not altered in the 0.30% Na group but significantly increased in the 0.04% Na group compared to the week 10 baseline (24h avg: +6±2 mmHg, p<0.05). Ganglionic block with chlorisondamine or β-adrenergic block with propranolol reduced heart rate (HR) to a greater extent (each p<0.05) in the 0.04% group. Conversely, muscarinic block with atropine resulted in a smaller increase in HR in this group (p<0.05). Urine elimination rates of aldosterone (0.30%: 1.96±0.44 vs 0.04%: 2.92±0.064 ng/d) and copeptin (0.30%: 0.16±0.03 vs. 0.04%: 0.14±0.02 ng/d) were similar between groups. Finally, at 18 weeks of age, all mice received the angiotensin AT 1 R antagonist losartan in drinking water. This caused a greater BP reduction in the 0.04% group at 20 weeks (24h avg: -12±2 vs -19±2 mmHg, p<0.05). Together, these data indicate that dietary Na restriction during hypothalamic development programs increased salt sensitivity of BP via a mechanism involving AT 1 R and characterized by increased sympathetic yet decreased parasympathetic activity.

Abstract 51: Targeted Apoptosis of the Angiotensin AT 1 R-Expressing Subtype of Agouti-Related Peptide Neurons Increases Resting Metabolism and Blood Pressure

Agouti-related peptide (AgRP) neurons in the hypothalamic arcuate nucleus (ARC) control feeding and resting metabolic rate (RMR) and may also contribute to blood pressure (BP) and heart rate (HR) control. Our recent studies demonstrate that three subtypes of ARC AgRP neurons can be distinguished by differential expression of angiotensin II (ANG) receptors and differential electrophysiological responses to ANG. The "Type 1" subtype of AgRP neurons in the ARC express the ANG type 1 receptor (AT 1 R), and application of ANG inhibits these cells via a Gi cascade, ultimately causing increased RMR. Other published data suggest that AT 1 R action in AgRP neurons may also stimulate BP. Thus, we hypothesized that cell-specific ablation of Type 1 AgRP neurons would disinhibit (increase) RMR and BP. Within the ARC, only Type 1 AgRP neurons express AT 1 R. Thus, cell-specific ablation of Type 1 AgRP neurons was achieved by injecting an Adeno-associated virus (AAV) encoding Cre-activatable Caspase3 into the ARC of adult AT 1 R CRE mice that express Cre-recombinase via the AT 1 R locus (n=10m+6f). Cre-deficient mice receiving AAV-Casp3 injection, mice receiving anesthesia but no virus, and AT 1 R CRE mice receiving an AAV vector encoding GFP were used as controls (n=21m+10f). Multiplexed metabolic phenotyping demonstrated that two weeks after injection, ablation of Type 1 cells grossly stimulated RMR (+11%: 0.331±0.007 vs. 0.366±0.008 kcal/h, p<0.01) without affecting activity-dependent expenditure; and paradoxically increased meal initiations (+10%, p<0.05) but reduced meal size (-14%, p<0.05), yielding no net change in total food intake after correction for body size and composition (+7%, p=0.24). Ablation of Type 1 neurons also increased preferred ambient temperature in a thermal gradient (29.4±0.3 vs 30.9±0.1 °C, p<0.01). In a separate cohort instrumented with radiotelemetric BP transducers (n=6m), HR progressively decreased (24h avg: -58±9 bpm, p<0.01) as systolic BP tended toward an increase (24h avg: +6±3 mmHg, p=0.10) through 5-7 weeks after ablation. We conclude that AT 1 R-expressing AgRP Type 1 neurons critically contribute to RMR control and appear to also influence BP/HR and feeding control. Therefore, we posit that altered function of these cells may mechanistically contribute to changes in RMR, BP/HR, and feeding control associated with obesity.

Functional consequences of spatial, temporal and ligand bias of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) regulate every aspect of kidney function by mediating the effects of various endogenous and exogenous substances. A key concept in GPCR function is biased signalling, whereby certain ligands may selectively activate specific pathways within the receptor's signalling repertoire. For example, different agonists may induce biased signalling by stabilizing distinct active receptor conformations - a concept that is supported by advances in structural biology. However, the processes underlying functional selectivity in receptor signalling are extremely complex, involving differences in subcellular compartmentalization and signalling dynamics. Importantly, the molecular mechanisms of spatiotemporal bias, particularly its connection to ligand binding kinetics, have been detailed for GPCRs critical to kidney function, such as the AT1 angiotensin receptor (AT1R), V2 vasopressin receptor (V2R) and the parathyroid hormone 1 receptor (PTH1R). This expanding insight into the multifaceted nature of biased signalling paves the way for innovative strategies for targeting GPCR functions; the development of novel biased agonists may represent advanced pharmacotherapeutic approaches to the treatment of kidney diseases and related systemic conditions, such as hypertension, diabetes and heart failure.

Valsartan Mitigates the Progression of Methotrexate-Induced Acute Kidney Injury in Rats via the Attenuation of Renal Inflammation and Oxidative Stress.

Methotrexate (MTX) is a folic acid antagonist, commonly administered for the treatment of a variety of cancers. However, methotrexate toxicity including bone marrow suppression and hepatic and renal toxicity limits its use. Angiotensin AT1 receptor blockers including Valsartan (Val) possess the ability to ameliorate MTX-induced toxicity through various mechanisms. In this study, we explored the potential reno-protective effects of Val against MTX-induced acute kidney injury in rats. Twenty-four Wistar rats were randomly segregated into 3 groups. Group 1 served as the control group and received an oral dose of 1mL/kg of normal saline. Group 2 received a single dose of 20 mg/kg of MTX intraperitoneally (IP) for 5 days. Group 3 received a single IP dose of 20 mg/kg of MTX followed by an oral dose of 10 mg/kg of Valsartan for 5 days. At the end of the experiment, the levels of serum kidney biomarkers, inflammatory and oxidative stress markers were accessed. Furthermore, the effect of MTX on kidney tissue histology was examined. Our results showed that MTX treatment increased the level of serum kidney and inflammatory biomarkers and decreased the level of antioxidants SOD and GSH while increasing the lipid peroxidation contents. Furthermore, MTX treatment caused structural changes to kidney histology. However, the administration of Val significantly prevented these changes. Valsartan possesses nephroprotective potential and might serve as a potential therapeutic strategy against MTX-induced kidney injury.

Effect of angiotensin II receptor blockade combined with a thiazide diuretic on plasma aldosterone escape in healthy subjects

Objective: The chronic administration of ARBs is associated with an incomplete ALDO suppression suggesting either a partial blockade of adrenal angiotensin AT1 receptors or a breakthrough phenomenon. We have investigated the adrenal response to exogenous Ang II in healthy subjects receiving an ARB alone or in association with a thiazide diuretic. Methods: Thirty subjects participated in this randomised, controlled, single-blind study. The effect of a 1 h infusion of Ang II (3 ng/kg/min) on plasma ALDO levels were investigated in each volunteer before and after a 1-week single-blind administration of either irbesartan 300 mg alone or in association with hydrochlorothiazide (HCTZ) 12.5 or 25 mg. After one week washout period, volunteers were switched to olmesartan 20mg/HCTZ 25 mg or valsartan 160/HCTZ 25 mg or losartan 100/HCTZ 25mg for another week. Results: Compared to pre-treatment values and to irbesartan alone, trough plasma ALDO values were higher in subjects receiving Irb300/12.5, and were significantly increased with Irb300/25 (to 4.9±1.8 and 7.5±1.2 ng/dl respectively, p<0.05). In participants receiving Olm20/25, Vals160/25 and Los100/25, trough ALDO were also significantly elevated. Before any treatment, exogenous Ang II increased plasma ALDO acutely from 3.6±0.7 to 13.2±1.3 ng/dl. With Irb300, the ALDO response to Ang II was blunted by 50-60% independently of the presence of HCTZ (p<0.05). The ALDO response to Ang II was also significantly blunted by about 50-60% with Olm20/25 and Vals160/25, but not with Los100/25. Conclusion: These results demonstrate that recommended doses of most ARBs only partially block the adrenal response to exogenous Ang II when associated with a thiazide diuretic. The ARB-thiazide association accentuates the aldosterone escape thus further justifying the use of mineralocorticoid antagonists in some clinical circumstances such as resistant hypertension or hypertension with severe end organ damage.

Transcriptomic profiling reveals a pronociceptive role for angiotensin II in inflammatory bowel disease.

Visceral pain is a leading cause of morbidity in inflammatory bowel disease (IBD), contributing significantly to reduced quality of life. Currently available analgesics often lack efficacy or have intolerable side effects, driving the need for a more complete understanding of the mechanisms causing pain. Whole transcriptome gene expression analysis was performed by bulk RNA sequencing of colonic biopsies from patients with ulcerative colitis (UC) and Crohn's disease (CD) reporting abdominal pain and compared with noninflamed control biopsies. Potential pronociceptive mediators were identified based on gene upregulation in IBD biopsy tissue and cognate receptor expression in murine colonic sensory neurons. Pronociceptive activity of identified mediators was assessed in assays of sensory neuron and colonic afferent activity. RNA sequencing analysis highlighted a 7.6-fold increase in the expression of angiotensinogen transcripts, Agt , which encode the precursor to angiotensin II (Ang II), in samples from UC patients ( P = 3.2 × 10 -8 ). Consistent with the marked expression of the angiotensin AT 1 receptor in colonic sensory neurons, Ang II elicited an increase in intracellular Ca 2+ in capsaicin-sensitive, voltage-gated sodium channel subtype Na V 1.8-positive sensory neurons. Ang II also evoked action potential discharge in high-threshold colonic nociceptors. These effects were inhibited by the AT 1 receptor antagonist valsartan. Findings from our study identify AT 1 receptor-mediated colonic nociceptor activation as a novel pathway of visceral nociception in patients with UC. This work highlights the potential utility of angiotensin receptor blockers, such as valsartan, as treatments for pain in IBD.

N-(Heteroaryl)thiophene sulfonamides as angiotensin AT2 receptor ligands

Two series of N-(heteroaryl)thiophene sulfonamides, encompassing either a methylene imidazole group or a tert-butylimidazolylacetyl group in the meta position of the benzene ring, have been synthesized. An AT2R selective ligand with a Ki of 42 nM was identified in the first series and in the second series, six AT2R selective ligands with significantly improved binding affinities and Ki values of <5 nM were discovered. The binding modes to AT2R were explored by docking calculations combined with molecular dynamics simulations. Although some of the high affinity ligands exhibited fair stability in human liver microsomes, comparable to that observed with C21 undergoing clinical trials, most ligands displayed a very low metabolic stability with t½ of less than 10 min in human liver microsomes. The most promising ligand, with an AT2R Ki value of 4.9 nM and with intermediate stability in human hepatocytes (t½ = 77 min) caused a concentration-dependent vasorelaxation of pre-contracted mouse aorta.

Antihypertensive treatment with hydrochlorothiazide-hydralazine combination aggravates medial vascular calcification in CKD rats with mineral bone disorder.

Arterial stiffness and medial vascular calcification, leading to isolated systolic blood pressure (BP), are major cardiovascular risk factors in patients with chronic kidney disease (CKD) and mineral bone disorders (MBD). The impact of BP on MBD-induced medial vascular calcification in CKD remains uncertain. We investigated whether BP reduction improves arterial stiffness and medial vascular calcification in a rat model of CKD-MBD. CKD was induced in Wistar rats by subtotal nephrectomy. Then, MBD was generated by a Ca/P-rich diet with calcitriol supplementation to induce medial vascular calcification. Two antihypertensive treatments were evaluated: (1) the angiotensin AT1 receptor antagonist losartan, and (2) the combination of the thiazide diuretic hydrochlorothiazide and the direct vasodilator hydralazine (HCTZ/HY). After 5 weeks, mean BP (MBP), pulse pressure (PP), and pulse wave velocity (PWV) were determined. Vascular calcification was assessed in the thoracic aorta. While MBP was similar in CKD-MBD and control CKD rats, PP and PWV were increased in CKD-MBD rats. The heightened arterial stiffness in CKD-MBD rats was associated with diffused medial calcification along the thoracic aorta. Although both losartan and HCTZ/HY reduced MBP in CKD-MBD rats, losartan did not affect PP and PWV nor medial vascular calcification, whereas HCTZ/HY, unexpectedly, further increased arterial stiffness and medial vascular calcification. In the rat model of CKD-MBD, antihypertensive treatment with losartan did not affect arterial stiffness or medial vascular calcification. However, HCTZ/HY treatment aggravated arterial stiffness and vascular calcification despite a similar reduction of MBP, suggesting a blood pressure-independent mechanism for vascular calcification.

Towards the convergent therapeutic potential of GPCRs in autism spectrum disorders.

Autism spectrum disorders (ASD) are diagnosed in 1/100 children worldwide, based on two core symptoms, deficits in social interaction and communication and stereotyped behaviours. G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors that transduce extracellular signals to convergent intracellular signalling and downstream cellular responses that are commonly dysregulated in ASD. Despite hundreds of GPCRs being expressed in the brain, only 23 are genetically associated with ASD according to the Simons Foundation Autism Research Initiative (SFARI) gene database: oxytocin OTR, vasopressin V1A , V1B , metabotropic glutamate mGlu5 , mGlu7 , GABAB2 , dopamine D1 , D2 , D3 , serotoninergic 5-HT1B , β2 -adrenoceptor, cholinergic M3 , adenosine A2A , A3 , angiotensin AT2 , cannabinoid CB1 , chemokine CX3 CR1, orphan GPR37, GPR85 and olfactory OR1C1, OR2M4, OR2T10, OR52M1. Here, we review the therapeutic potential of these 23 GPCRs, 5-HT2A and 5-HT7 for ASD. For each GPCR, we discuss its genetic association, genetic and pharmacological manipulation in animal models, pharmacopeia for core symptoms of ASD and rank them based on these factors. Among these GPCRs, we highlight that D2 , 5-HT2A , CB1 , OTR and V1A are the most promising targets for ASD. We discuss that the dysregulation of GPCRs and their signalling is a convergent pathological mechanism of ASD. Their therapeutic potential has only begun as multiple GPCRs could mitigate ASD.

Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT2 receptor ligands reveals that EMA401 and PD123319 have agonistic properties

With the discovery of the protective arm of the renin-angiotensin system (RAS), interest has grown in protective RAS-related receptors such as the angiotensin AT2-receptor [AT2R] as potential new drug targets. While it is known that AT2R couple to Gi, it is also apparent that they do not signal via inhibition of adenylyl cyclase/decrease in cAMP, as do many Gi-coupled receptors. Thus, standard commercially-available assays cannot be applied to test for agonistic or antagonistic properties of AT2R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT2R.Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT2R ligands, primarily for distinguishing between AT2R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT2R-transfected CHO cells is a reliable assay for the characterization of AT2R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT2R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT2R-transfected HEK-293 cells are unresponsive to AT2R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT2R antagonists, are partial agonists at the AT2R.Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT2R-CHO cells that is suitable for the characterisation of novel and established AT2R ligands.

Frequency-coded patterns of sympathetic vasomotor activity are differentially evoked by the paraventricular nucleus of the hypothalamus in the Goldblatt hypertension model.

The paraventricular nucleus of the hypothalamus (PVN) contains premotor neurons involved in the control of sympathetic vasomotor activity. It is known that the stimulation of specific areas of the PVN can lead to distinct response patterns at different target territories. The underlying mechanisms, however, are still unclear. Recent evidence from sympathetic nerve recording suggests that relevant information is coded in the power distribution of the signal along the frequency range. In the present study, we addressed the hypothesis that the PVN is capable of organizing specific spectral patterns of sympathetic vasomotor activation to distinct territories in both normal and hypertensive animals. To test it, we investigated the territorially differential changes in the frequency parameters of the renal and splanchnic sympathetic nerve activity (rSNA and sSNA, respectively), before and after disinhibition of the PVN by bicuculline microinjection. Subjects were control and Goldblatt rats, a sympathetic overactivity-characterized model of neurogenic hypertension (2K1C). Additionally, considering the importance of angiotensin II type 1 receptors (AT1) in the sympathetic responses triggered by bicuculline in the PVN, we also investigated the impact of angiotensin AT1 receptors blockade in the spectral features of the rSNA and sSNA activity. The results revealed that each nerve activity (renal and splanchnic) presents its own electrophysiological pattern of frequency-coded rhythm in each group (control, 2K1C, and 2K1C treated with AT1 antagonist losartan) in basal condition and after bicuculline microinjection, but with no significant differences regarding total power comparison among groups. Additionally, the losartan 2K1C treated group showed no decrease in the hypertensive response triggered by bicuculline when compared to the non-treated 2K1C group. However, their spectral patterns of sympathetic nerve activity were different from the other two groups (control and 2K1C), suggesting that the blockade of AT1 receptors does not totally recover the basal levels of neither the autonomic responses nor the electrophysiological patterns in Goldblatt rats, but act on their spectral frequency distribution. The results suggest that the differential responses evoked by the PVN were preferentially coded in frequency, but not in the global power of the vasomotor sympathetic responses, indicating that the PVN is able to independently control the frequency and the power of sympathetic discharges to different territories.

Telmisartan and losartan: The marked differences between their chemical and pharmacological properties may explain the difference in therapeutic efficacy in hospitalized patients with COVID‐19

Telmisartan and losartan: The marked differences between their chemical and pharmacological properties may explain the difference in therapeutic efficacy in hospitalized patients with COVID‐19

NaHS restores the vascular alterations in the renin-angiotensin system induced by hyperglycemia in rats

Hyperglycemia (HG) impairs the renin-angiotensin system (RAS), which may contribute to vascular dysfunction. Besides, hydrogen sulfide (H2S) exerts beneficial cardiovascular effects in metabolic diseases. Therefore, our study aimed to determine the effects of chronic administration of sodium hydrosulfide (NaHS; inorganic H2S donor) and DL-Propargylglycine [DL-PAG; cystathionine-ץ-lyase (CSE) inhibitor] on the RAS-mediated vascular responses impairments observed in thoracic aortas from male diabetic Wistar rats. For that purpose, neonatal rats were divided into two groups that received: 1) citrate buffer (n = 12) or 2) streptozotocin (STZ, 70 mg/kg; n = 48) on the third postnatal day. After 12 weeks, diabetic animals were divided into 4 subgroups (n = 12 each) that received daily i.p. injections during 4 weeks of: 1) non-treatment; 2) vehicle (PBS, 1 mL/kg); 3) NaHS (5.6 mg/kg); and 4) DL-PAG (10 mg/kg). After treatments (16 weeks), blood glucose, angiotensin-(1−7) [Ang-(1−7)], and angiotensin II (Ang II) levels, vascular responses to Ang-(1−7) and Ang II, and the expression of angiotensin AT1, AT2, and Mas receptors, angiotensin converting enzyme (ACE) and ACE type 2 (ACE2) were determined. HG induced: 1) increased blood glucose levels and expression of angiotensin II AT1 receptor; 2) impaired Ang-(1−7) and Ang II mediated vascular responses; 3) decreased angiotensin levels and expression of angiotensin II AT2 and angiotensin-(1−7) Mas receptors, and ACE2; and 4) no changes in ACE expression. Interestingly, NaHS, but not DL-PAG, reversed HG-induced impairments, except for blood glucose level changes. These results suggest that NaHS restores vascular function in streptozotocin-induced HG through RAS modulation.

The cannabinoid CB1 receptor interacts with the angiotensin AT2 receptor. Overexpression of AT2-CB1 receptor heteromers in the striatum of 6-hydroxydopamine hemilesioned rats

It is of particular interest the potential of cannabinoid and angiotensin receptors as targets in the therapy of Parkinson's disease (PD). While endocannabinoids are neuromodulators that act through the CB1 and CB2 cannabinoid receptors, the renin angiotensin-system is relevant for regulation of the correct functioning of several brain circuits. Resonance energy transfer assays in a heterologous system showed that the CB1 receptor (CB1R) can directly interact with the angiotensin AT2 receptor (AT2R). Coactivation of the two receptors results in increased Gi-signaling. The AT2-CB1 receptor heteromer imprint consists of a blockade of AT2R-mediated signaling by rimonabant, a CB1R antagonist. Interestingly, the heteromer imprint, discovered in the heterologous system, was also found in primary striatal neurons thus demonstrating the expression of the heteromer in these cells. In situ proximity ligation assays confirmed the occurrence of AT2-CB1 receptor heteromers in striatal neurons. In addition, increased expression of the AT2-CB1 receptor heteromeric complexes was detected in the striatum of a rodent PD model consisting of rats hemilesioned using 6-hydroxydopamine. Expression of the heteromer was upregulated in the striatum of lesioned animals and, also, of lesioned animals that upon levodopa treatment became dyskinetic. In contrast, there was no upregulation in the striatum of lesioned rats that did not become dyskinetic upon chronic levodopa treatment. The results suggest that therapeutic developments focused on the CB1R should consider that this receptor can interact with the AT2R, which in the CNS is involved in mechanisms related to addictive behaviors and to neurodegenerative and neuroinflammatory diseases.

The effect of daidzein on renal injury in ovariectomized rats: interaction of angiotensin receptors and long non-coding RNAs H19, GAS5, MIAT, and Rian.

Renin-angiotensin system (RAS) is prominently associated with renal pathophysiology in postmenopausal women. Long non-coding RNAs (lncRNAs) H19, GAS5, MIAT, and Rian have been linked to the pathogenesis of renal injury. This study aimed to evaluate the beneficial effects of daidzein on unilateral ureteral obstruction (UUO) induced-renal injury in ovariectomized (OVX) rats through interaction with angiotensin AT1, Mas receptors, and lncRNAs. 84 female rats were ovariectomized (OVX) two weeks before performing obstruction of the left kidney ureter (UUO). The animals were then randomly divided into four main groups (n=21): Sham+DMSO, UUO+DMSO, UUO+17β-Estradiol (E2) (positive control), and UUO+daidzein. Each main group comprised three subgroups (n=7) and were treated with saline, A779 (MasR antagonist), or losartan (AT1R antagonist) for 15 days. On day 16, the animals were euthanized, and the left kidneys were harvested for histopathology and lncRNAs expression assays. UUO significantly increased kidney tissue damage score (KTDS) in the UUO rats, increased the expression of H19 and MIAT, and decreased the expression of GAS5 and Rian. Daidzein alone and in co-treatment with losartan or A779 reversed these effects. Daidzein with 1 mg/kg dose was more effective than E2. Daidzein alone and in co-treatment with A779 and losartan improved renal injury in UUO rats and recovered dysregulated expression of UUO-related lncRNAs through modulating MasR and AT1R receptors, associating with modulation of the expression of lncRNAs. Daidzein could be considered a renoprotective phytoestrogen substitute for E2 therapy in postmenopausal women suffering from renal diseases.

S-12-7: THE RENIN ANGIOTENSIN SYSTEM IN BASIC AND CLINICAL RESEARCH

The renin-angiotensin-system (RAS) is one of the oldest known hormonal systems preserved in multiple species over millions of years. A key enzyme of the system, renin, was discovered in 1898, and ever since a huge body of research has continuously revealed physiological and pathophysiological features of the RAS. Classically, these include, via the so-called angiotensin AT1 receptor, powerful vasoconstriction, regulation of the body fluid balance by inducing thirst, stimulation of aldosterone release from the adrenal cortex, renal salt retention, stimulation of the sympathetic nervous system and, more recently discovered, pro-fibrotic and pro-inflammatory effects. Pathophysiological actions of the RAS, with its extension to those of aldosterone (RAAS), include the induction of arterial hypertension, cardiac and vascular hypertrophy, lung and renal fibrosis, vascular atherosclerosis, perivascular fibrosis or chronic inflammation and others. Attempts to inhibit the RAS in hypertension and other cardiovascular diseases first led to the clinical introduction of ACE-inhibitors some forty years ago, followed by selective antagonists of the AT1 receptor, the sartans. After a multitude of evidence-generating trials and global success in clinical practice, both approaches are now firmly established in the therapy of hypertension as well as cardio-metabolic and renal diseases. More recently, research has focused on the so-called protective arm of the RAS, mediated by stimulation of the angiotensin AT2 receptor by or by the Mas receptor stimulated by the angiotensin 1–7 peptide. Activation of these systems has exhibited beneficial effects in numerous preclinical studies via opposing AT1-mediated actions and beyond. Other degradation peptides of the angiotensin precursor, angiotensinogen, may also exhibit protective effects via these receptors. Agonists for the AT2 receptor as well as for the ACE2-Mas system have been developed and are currently under clinical evaluation. It remains to be seen whether the pharmacological stimulation of the protective arm of the RAS will become an effective clinical tool in various cardiovascular and metabolic indications. Theoretically, it would make sense not only to block the harmful RAS but, at the same time, also stimulate the beneficial actions of the system.

Effect of the renin-angiotensin system on the exacerbation of adrenal glucocorticoid steroidogenesis in diabetic mice: Role of angiotensin-II type 2 receptor.

Prior investigation shows an increase in the activity of both hypothalamus-pituitary-adrenal (HPA) axis and the renin-angiotensin system (RAS) in diabetic patients. Moreover, activation of angiotensin-II type 1 receptor (AT1) has been associated with adrenal steroidogenesis. This study investigates the role of RAS on the overproduction of corticosterone in diabetic mice. Diabetes was induced by intravenous injection of alloxan into fasted Swiss-webster mice. Captopril (angiotensin-converting enzyme inhibitor), Olmesartan (AT1 receptor antagonist), CGP42112A (AT2 receptor agonist) or PD123319 (AT2 receptor antagonist) were administered daily for 14 consecutive days, starting 7 days post-alloxan. Plasma corticosterone was evaluated by ELISA, while adrenal gland expressions of AT1 receptor, AT2 receptor, adrenocorticotropic hormone receptor MC2R, pro-steroidogenic enzymes steroidogenic acute regulatory protein (StAR), and 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1) were assessed using immunohistochemistry or western blot. Diabetic mice showed adrenal gland overexpression of AT1 receptor, MC2R, StAR, and 11βHSD1 without altering AT2 receptor levels, all of which were sensitive to Captopril or Olmesartan treatment. In addition, PD123319 blocked the ability of Olmesartan to reduce plasma corticosterone levels in diabetic mice. Furthermore, CGP42112A significantly decreased circulating corticosterone levels in diabetic mice, without altering the overexpression of MC2R and StAR in the adrenal glands. Our findings revealed that inhibition of both angiotensin synthesis and AT1 receptor activity reduced the high production of corticosterone in diabetic mice via the reduction of MC2R signaling expression in the adrenal gland. Furthermore, the protective effect of Olmesartan on the overproduction of corticosterone by adrenals in diabetic mice depends on both AT1 receptor blockade and AT2 receptor activation.

Quantitative Phosphoproteomics of the Angiotensin AT2-Receptor Signaling Network Identifies HDAC1 (Histone-Deacetylase-1) and p53 as Mediators of Antiproliferation and Apoptosis.

Angiotensin AT2-receptor signaling is atypical for a G-protein coupled receptor and incompletely understood. To obtain novel insights into AT2-receptor signaling, we mapped changes in the phosphorylation status of the entire proteome of human aortic endothelial cells in response to AT2-receptor stimulation. Phosphorylation status of human aortic endothelial cells after stimulation with C21 (1 µM; 0, 1, 3, 5, 20 minutes) was determined utilizing time-resolved quantitative phosphoproteomics. Specific changes in protein phosphorylation and acetylation were confirmed by Western Blotting. Functional tests included resazurin assay for cell proliferation, and caspase 3/7 luminescence assay or FACS analysis of annexin V expression for apoptosis. AT2-receptor stimulation significantly altered the phosphorylation status of 172 proteins (46% phosphorylations, 54% dephosphorylations). Bioinformatic analysis revealed a cluster of phospho-modified proteins involved in antiproliferation and apoptosis. Among these proteins, HDAC1 (histone-deacetylase-1) was dephosphorylated at serine421/423 involving serine/threonine phosphatases. Resulting HDAC1 inhibition led to p53 acetylation and activation. AT2-receptor stimulation induced antiproliferation and apoptosis, which were absent when cells were co-incubated with the p53 inhibitor pifithrin-α, thus indicating p53-dependence of these AT2-receptor mediated functions. Contrary to the prevailing view that AT2-receptor signaling largely involves phosphatases, our study revealed significant involvement of kinases. HDAC1 inhibition and resulting p53 activation were identified as novel, AT2-receptor coupled signaling mechanisms. Furthermore, the study created an openly available dataset of AT2-receptor induced phospho-modified proteins, which has the potential to be the basis for further discoveries of currently unknown, AT2-receptor coupled signaling mechanisms.

Isoflavone daidzein ameliorates renal dysfunction and fibrosis in a postmenopausal rat model: Intermediation of angiotensin AT1 and Mas receptors and microRNAs 33a and 27a.

Chronic kidney disease (CKD), accompanied by renal dysfunction, fibrosis, and apoptosis, is highly prevalent in postmenopausal women. We tested the hypothesis that isoflavone daidzein may ameliorate renal dysfunction and fibrosis through angiotensin II type 1 (AT1R) and angiotensin 1-7 (MasR) receptors in association with microRNAs 33a and 27a. Two weeks before the initiation of the experiments, rats (n=84) underwent ovariectomy (OVX). Then, unilateral ureteral obstruction (UUO) was performed in OVX rats, and animals were allocated to the following groups (n=21): sham vehicle (dimethyl sulfoxide; DMSO 1%), UUO vehicle, UUO+17β-estradiol (E2), and UUO+daidzein. Each group encompassed three subgroups (n=7) treated with saline, A779 (MasR antagonist), or losartan (AT1R antagonist) for 15 days. The fractional urine excretion of sodium (FENa+) and potassium (FEK+), renal failure index (RFI), renal interstitial fibrosis (RIF index), glomerulosclerosis, miR-33a, and miR-27a expressions and their target genes were analyzed. Apoptosis was measured via cleaved caspase-3 immunohistochemistry. UUO increased kidney weight, FENa+, FEK+, urine calcium, RFI, RIF index, glomerulosclerosis, and cleaved caspase-3. Moreover, expression of renal miR-33a and miR-27a, collagen3A1 mRNA, and protein were up-regulated post-UUO. Daidzein treatment alleviated the harmful effects of UUO especially in co-treatment with losartan. They also masked the anticipated worsening effects of A779 on UUO. Compared with E2, daidzein efficiently ameliorated renal dysfunction, fibrosis, and apoptosis through modulation of miR-33a and miR-27a expression and their crosstalk with AT1R and MasR. Therefore, daidzein might be a promising candidate for treating CKD in postmenopausal and older women.