Elevated Angiotensin II Research Articles

ANGIOTENSIN-(1-7) FORMATION UPON FLUID FLOW SHEAR STRESS IN HUMAN PODOCYTES

Objective: Glomerular hyperfiltration associates with increased fluid flow shear stress (FFSS) on podocytes. This can lead to irreversible podocyte damage, which in turn negatively affects the permeability and integrity of the glomerular filtration barrier. AngiotensinII (AngII) is an important mediator in chronic kidney disease. The negative effects of AngII are at least partially opposed by the alternative signaling pathways within the renin-angiotensin system (RAS) consisting of angiotensin-converting enzyme 2 (ACE2), angiotensin-(1-7) (Ang-[1-7]) and the MAS1 receptor. This counteracting pathway may exert renoprotective effects and support anti-inflammatory processes. As the pathomechanisms and signaling pathways underlying podocyte damage induced by FFSS are not fully understood, our aim is to elucidate the potential renoprotective impact of the alternative RAS axis in human podocytes in the context of glomerular hyperfiltration. Design and method: Conditionally immortalized human podocytes (hPCs) were used for mechanistic studies. Cells seeded on collagen IV coated Culture Slips® were inserted in a Streamer® Shear Stress Device and exposed to FFSS of 1dyne/cm2 for 2h. Control cells were not exposed to FFSS. Afterwards, the formation rate of AngII to Ang-[1-7] was measured in whole cell lysates (1 experiment, n = 6 samples per condition) using spiked AngII as a substrate with and without addition of Z-Pro-Prolinal and MLN-4760. Additionally, gene expression analyses of the enzymes involved in Ang-[1-7] formation were performed. Results: Preliminary data show an increased AngII to Ang-[1-7] formation rate in hPCs following FFSS exposure compared to control. Ang-[1-7] formation under both conditions is reduced by addition of Z-Pro-Prolinal, whereas MLN-4760 has no effect. RNA-expression levels of prolylcarboxypeptidase and prolylendopeptidase are not significantly affected upon FFSS compared to control. In contrast, ACE2 is significantly upregulated by FFSS although expression levels are substantially lower under both conditions compared to prolylcarboxypeptidase and prolylendopeptidase. Conclusions: Our results suggest an elevated AngII to Ang-[1-7] formation rate upon FFSS in hPCs compared to no-flow control. Under both settings, Ang-[1-7] formation in hPCs seems to be mediated by prolylcarboxypeptidase and/or prolylendopeptidase but not ACE2. These results support further studies to confirm the potential role and mechanisms of Ang-[1-7] upregulation in response to glomerular hyperfiltration.

Chronic angiotensin II signaling drives sustained PIP2 deficits and altered sarcolemmal Cav1.2 expression and function in cardiomyocytes.

Contractile dysfunction, hypertrophy, and cell death during heart failure are linked to altered Ca2+ handling and elevated levels of angiotensin II (AngII). This hormone signals through Gq-coupled AT1 receptors, initiating hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). Previous work revealed acute AngII signaling and PIP2 depletion destabilizes CaV1.2 clusters in cardiomyocytes, triggering their internalization, and subsequent reduction of whole-cell Ca2+ current (ICa) and contractility. However, the effects of chronically elevated AngII on the phospholipid landscape and associated effects on ICa remain unknown. We subjected mice to sustained delivery of AngII to test the impact on phosphoinositides, ICa, and excitation-contraction coupling. We hypothesized that chronic AngII signaling leads to sustained PIP2 and CaV1.2 deficits, contributing to the diminished functional output associated with disease development. Phospholipid mass spectrometry revealed extensive alteration of phosphoinositide species, including a ∼44% reduction in PIP2 and ∼37% reduction in cardioprotective PIP3 in AngII-infused hearts compared to controls. To study the effects of this phospholipid imbalance on CaV1.2, we examined ICa, finding similar current density in both groups. However, super-resolution imaging revealed a ∼54% reduction in t-tubular CaV1.2 expression and a ∼23% reduction in cluster area. Conserved ICa density despite reduced t-tubular CaV1.2 suggests that either the channels have relocated to the sarcolemmal crest, or their Po has increased. We examined crest CaV1.2 and observed no change with AngII, thus the Po of the remaining channels must increase to maintain ICa. Western blots revealed enhanced phosphorylation of CaV1.2, phosphorylation of RyR2, and an increased expression of the catalytic subunit of PKA, suggesting a compensatory sympathetic activation underlying conserved ICa. These findings support a model wherein chronic elevation of AngII disrupts phosphoinositide and CaV1.2 levels, elucidating the role of chronic AngII in disease progression.

Elevated Angiotensin-II Levels Contribute to the Pathogenesis of Open-Angle Glaucoma Via Inducing the Expression of Fibrosis-Related Genes in Trabecular Meshwork Cells Through a ROS/NOX4/SMAD3 Axis

Glaucoma including primary open-angle glaucoma (POAG) results from elevations in intraocular pressure (IOP). An eye-localized renin-angiotensin system (RAS) has been implicated in IOP regulation, although its mechanism of action and contribution to glaucoma is poorly understood. Here, we detected significant increases in the levels of angiotensin II (ANGII) in aqueous humor samples from POAG patients. Moreover, we determined that the concentrations of ANGII were positively correlated with IOP, suggesting a role for elevated ANGII levels in eye pathogenesis. Functional investigations demonstrated that ANGII induces the expression of fibrosis-related genes of transformed and primary human trabecular meshwork cells (HTMCs) through the transcriptional upregulation of key fibrotic genes. Parallel experiments using a murine periocular conjunctival fornix injection model confirmed that ANGII induces the expression of fibrosis-related genes in trabecular meshwork (TM) cells in vivo along with increasing IOP. ANGII was revealed to function through increasing the levels of reactive oxygen species (ROS) via selectively upregulating NOX4, with NOX4 knockdown or inhibition with GLX351322 alleviating fibrotic changes induced by ANGII. We further show that ANGII activates Smad3, with both GLX351322 and an inhibitor of Smad3 (SIS3) decreasing the phosphorylation of Smad3 and dampening the ANGII-induced increases in fibrotic proteins. Moreover, NOX4 and Smad3 inhibitors also partially rescued the elevated IOP levels induced by ANGII. Our collective results therefore highlight ANGII as a biomarker and treatment target in POAG together with establishing a causal relationship between ANGII and up-regulation of the expression of fibrosis-related genes of TM cells via a NOX4/ROS axis in cooperation with TGFβ/Smad3 signaling.

Soluble ACE2 Is Filtered into the Urine.

ACE2 is a key enzyme in the renin-angiotensin system (RAS) capable of balancing the RAS by metabolizing angiotensin II (AngII). First described in cardiac tissue, abundance of ACE2 is highest in the kidney, and it is also expressed in several extrarenal tissues. Previously, we reported an association between enhanced susceptibility to hypertension and elevated renal AngII levels in global ACE2-knockout mice. To examine the effect of ACE2 expressed in the kidney, relative to extrarenal expression, on the development of hypertension, we used a kidney crosstransplantation strategy with ACE2-KO and WT mice. In this model, both native kidneys are removed and renal function is provided entirely by the transplanted kidney, such that four experimental groups with restricted ACE2 expression are generated: WT→WT (WT), KO→WT (KidneyKO), WT→KO (SystemicKO), and KO→KO (TotalKO). Additionally, we used nanoscale mass spectrometry-based proteomics to identify ACE2 fragments in early glomerular filtrate of mice. Although significant differences in BP were not detected, a major finding of our study is that shed or soluble ACE2 (sACE2) was present in urine of KidneyKO mice that lack renal ACE2 expression. Detection of sACE2 in the urine of KidneyKO mice during AngII-mediated hypertension suggests that sACE2 originating from extrarenal tissues can reach the kidney and be excreted in urine. To confirm glomerular filtration of ACE2, we used micropuncture and nanoscale proteomics to detect peptides derived from ACE2 in the Bowman's space. Our findings suggest that both systemic and renal tissues may contribute to sACE2 in urine, identifying the kidney as a major site for ACE2 actions. Moreover, filtration of sACE2 into the lumen of the nephron may contribute to the pathophysiology of kidney diseases characterized by disruption of the glomerular filtration barrier.

High salt induced augmentation of angiotensin II mediated hypertension is associated with differential expression of tumor necrosis factor-alpha receptors in the kidney

Aim: Chronic high salt (HS) intake causes minimal changes in blood pressure (BP) but it induces augmented hypertensive response to angiotensin II (AngII) administration in rodents. The mechanism of this augmentation is not clearly understood. As tumor necrosis factor-alpha (TNF-α) induces natriuresis by activating TNF-α receptor type 1 (TNFR1) but not type 2 (TNFR2), we hypothesize that TNFR1 activity is reduced when HS is given in combination of AngII that leads to enhanced sodium retention and thus, causing augmented hypertension. The aim of this study is to examine the responses to chronic HS intake and AngII administration on the renal tissue protein expressions of TNFR1 and TNFR2 in mice. Methods: Different groups of mice (n = 6–7 in each group) chronically treated with or without AngII (25 ng/min; implanted minipump) for 4 weeks which were fed either normal salt (NS; 0.4% NaCl) or high salt (HS; 4% NaCl) diets. Systemic BP was measured by tail-cuff plethysmography. At the end of treatment period, kidneys were harvested after sacrificing the mice with euthanasia. Immuno-histochemical analysis of TNFR1 and TNFR2 proteins in renal tissues was performed by measuring the staining area and the intensity of receptors’ immunoreactivities using NIS-Elements software. The results were expressed in percent area of positive staining and the relative intensity. Results: HS intake alone did not alter mean BP (HS; 77 ± 1 vs. NS; 76 ± 3 vs. mmHg; tail-cuff plethysmography) but AngII induced increases in BP were augmented in HS group (104 ± 2 vs. 95 ± 2 mmHg; P < 0.05). The area of TNFR1 staining was higher in HS than NS group (6.0 ± 0.9% vs. 3.2 ± 0.7%; P < 0.05) but it was lower in AngII + HS than in AngII + NS group (5.0 ± 0.7% vs. 6.3 ± 0.7%; P = 0.068). TNFR2 immunoreactivity was minimal in NS and HS groups but it was high in AngII + NS and even higher in AngII + HS group. Conclusions: These data suggest that the HS induced increased TNFR1 activity that facilitates enhanced sodium excretion is compromised in elevated AngII condition leading to salt retention and augmented hypertension.

Angiotensin II-induced renal angiotensinogen formation is enhanced in mice lacking tumor necrosis factor-alpha type 1 receptor.

In hypertension induced by angiotensin II (AngII) administration with high salt (HS) intake, intrarenal angiotensinogen (AGT) and tumor necrosis factor‐alpha (TNF‐α) levels increase. However, TNF‐α has been shown to suppress AGT formation in cultured renal proximal tubular cells. We examined the hypothesis that elevated AngII levels during HS intake reduces TNF‐α receptor type 1 (TNFR1) activity in the kidneys, thus facilitating increased intrarenal AGT formation. The responses to HS diet (4% NaCl) with chronic infusion of AngII (25 ng/min) via implanted minipump for 4 weeks were assessed in wild‐type (WT) and knockout (KO) mice lacking TNFR1 or TNFR2 receptors. Blood pressure was measured by tail‐cuff plethysmography, and 24‐h urine samples were collected using metabolic cages prior to start (0 day) and at the end of 2nd and 4th week periods. The urinary excretion rate of AGT (uAGT; marker for intrarenal AGT) was measured using ELISA. HS +AngII treatment for 4 weeks increased mean arterial pressure (MAP) in all strains of mice. However, the increase in MAP in TNFR1KO (77 ± 2 to 115 ± 3 mmHg; n = 7) was significantly greater (p < 0.01) than in WT (76 ± 1 to 102 ± 2 mmHg; n = 7) or in TNFR2KO (78 ± 2 to 99 ± 5 mmHg; n = 6). The increase in uAGT at 4th week was also greater (p < 0.05) in TNFR1KO mice (6 ± 2 to 167 ± 75 ng/24 h) than that in WT (6 ± 3 to 46 ± 16 ng/24 h) or in TNFR2KO mice (8 ± 7 to 65 ± 44 ng/24 h). The results indicate that TNFR1 exerts a protective role by mitigating intrarenal AGT formation induced by elevated AngII and HS intake.

Angiotensin II-induced natriuresis is attenuated in knockout mice lacking the receptors for tumor necrosis factor-α.

Intravenous infusion of relatively higher doses of angiotensin II (AngII) elicits natriuresis as opposed to its usual anti‐natruretic response. As AngII can induce tumor necrosis factor‐α (TNFα) production which elicits natriuresis via its action on TNFα receptor type 1 (TNFR1), we hypothesize that the concomitant release of TNFα contributes to the natriuretic response to AngII. Responses to AngII infusion (1 ng min−1 g−1 for 75 min, iv) were evaluated in anesthetized knockout (KO) mice lacking TNFR1 (n = 6) and TNFR2 (TNFα receptor type 2; n = 6) and compared these responses with those in wild type (WT; n = 6) mice. Arterial pressure (AP) was recorded from a cannula placed in the carotid artery. Renal blood flow (RBF) and glomerular filtration rate (GFR) were measured by PAH and inulin clearances, respectively. Urine was collected from a catheter placed in the bladder. AngII caused similar increases (p < 0.05 vs basal values) in AP (WT, 37 ± 5%; TNFR1KO, 35 ± 4%; TNFR2KO, 30 ± 4%) and decreases (p < 0.05) in RBF (WT, −39 ± 5%; TNFR1KO, −28 ± 6%; TNFR2KO, −31 ± 4%) without significant changes in GFR (WT, −17 ± 7%; TNFR1KO, −18 ± 7%; TNFR2KO, −12 ± 7%). However, despite similar changes in AP and renal hemodynamics, AngII induced increases (p < 0.05) in urinary sodium excretion in WT (3916 ± 942%) were less in the KO strains, more or less in TNFR1KO (473 ± 170%) than in TNFR2KO (1176 ± 168%). These data indicate that TNF‐α receptors, particularly TNFR1 are involved in the natriuretic response that occur during acute infusion of AngII and thus, plays a protective role in preventing excessive salt retention at clinical conditions associated with elevated AngII level.

Uncoupling Sickle Hematopoiesis Induced Angiotensin Receptor Signaling in Sickle Nephropathy

Angiotensin II (Ang-II) is a major bioactive peptide of the renin-angiotensin system. We recently reported hyperangiotensinemia in mice and patients with sickle cell disease (SCD), likely mediated by SCD-associated vascular damage (Chang, et al Nat Commun 2015). Herein, we explored whether the hyperangiotensinemia was driven by sickle hematopoiesis and its role in sickle nephropathy. We transplanted bone marrow from Berkeley sickle mice (SS) into C57Bl/6 WT mice (WT) and followed mice fully chimeric for sickle hematopoiesis for 6-9 months. Control WT mice were transplanted with bone marrow from WT mice. We observed that the SS/WT sickle hematopoietic chimeras developed elevated plasma Ang-II levels while the control chimeras did not, suggesting that sickle hematopoietic cells instigate hyperangiotensinemia. We then placed SS/WT chimeric mice or untransplanted Berk-SS mice on captopril, an angiotensin converting enzyme inhibitor (ACE-inhibitor) that would lower plasma Ang-II levels, and as expected Ang-II levels returned to baseline levels found in control mice. Additionally, an improvement in albuminuria and focal segmental glomerulo-necrosis (FSGN) was also noted, implying that sickle glomerulopathy is dependent on sickle hematopoiesis driven hyperangiotensinemia, and blocking this signaling can reverse the glomerular pathology. Similar results were also seen with the use of losartan, an Ang-II receptor 1 blocker (AT1R blocker) in SS/WT chimeras and untransplanted Berk-SS mice. This finding was confirmed by transplanting Berk-SS bone marrow in AT1R knockout mice (SS/AT1R-/- chimeras). Ang-II-AT1R signaling resulted in increased Smad1/2 phosphorylation, nitrotyrosine and TGFβ1 production in SS/WT glomeruli, resulting in FSGS that was absent in SS/AT1R-/- mice. While AT1R blockade improved glomerular pathology and albuminuria, the urine concentration defect (hyposthenuria), present in SCD, worsened with AT1R blockade with losartan. Hyposthenuria was even more severe in captropril treated Berk-SS mice, resulting in high mortality, which was averted only if they received supplemental hydration. Hence, while increased Ang-II signaling caused sickle glomerulopathy, Ang-II protected against severe hyposthenuria instigated by SCD, by signaling through AT1R and likely through a second receptor, conceivably AT2R. AT2R is known to have renoprotective functions, and AT2R-/- mice develop worse experimentally induced acute tubular necrosis. We investigated the mechanism of induction of hyposthenuria further using mice deficient in both Ang-II receptors and generating SS/AT1R-/- and SS/AT2R-/- chimeras along with the appropriate controls (WT/AT1R-/-, WT/AT2R-/-, WT/WT and SS/WT chimeric mice), by transplanting bone marrow from Berk-SS or WT mice into WT, AT1R-/- and AT2R-/- mice. Dual AT1R and AT2R deficiency is embryonic lethal. WT/WT mice had no hyposthenuria, while SS/WT chimeras developed the classic hyposthenuria associated with SCD. In AT1R chimeras: Both WT/AT1R-/- and SS/AT1R-/- chimeras developed hyposthenuria compared to WT/WT mice, demonstrating that Ang-II-AT1R signaling helps concentrate urine both in normal and sickle mice. In AT2R chimeras: WT/AT2R-/- chimeras had no hyposthenuria as WT/WT chimeras, suggesting that AT2R plays little role in concentrating urine in normal situations. However, SS/AT2R-/- chimeras developed significantly worse hyposthenuria than SS/WT chimeras, suggesting that under the stress of sickle cell disease, Ang-II-AT2R signaling improves hyposthenuria caused by SCD. Hence, while increased Ang-II signaling causes glomerulopathy via AT1R, it improves urine concentration in SCD both via AT1R and AT2R signaling. Indeed, a combination of AT1R inhibition with losartan, and an AT2R agonism via C21 (kindly provided by Vicore Pharma) improved both albuminuria and hyposthenuria in sickle mice. In summary, we show the molecular basis of sickle nephropathy and demonstrates that sickle hematopoiesis induces pathologic hyperangiotensinemia; increased Ang-II-AT1R signaling leads to glomerulopathy and end stage renal disease, despite its protective role in urine concentration; Ang-II-AT2R signaling improves hyposthenuria. Therefore, clinically, reduced AT1R signaling can improve sickle nephropathy, and dual AT1R blockade with AT2R agonism can be explored as a preferred therapeutic target. DisclosuresNo relevant conflicts of interest to declare.

Integration of Hypernatremia and Angiotensin II by the Organum Vasculosum of the Lamina Terminalis Regulates Thirst.

The organum vasculosum of the lamina terminalis (OVLT) contains NaCl-sensitive neurons to regulate thirst, neuroendocrine function, and autonomic outflow. The OVLT also expresses the angiotensin II (AngII) type1 receptor, and AngII increases Fos expression in OVLT neurons. The present study tested whether individual OVLT neurons sensed both NaCl and AngII to regulate thirst and body fluid homeostasis. A multifaceted approach, including in vitro whole-cell patch recordings, in vivo single-unit recordings, and optogenetic manipulation of OVLT neurons, was used in adult, male Sprague Dawley rats. First, acute intravenous infusion of hypertonic NaCl or AngII produced anatomically distinct patterns of Fos-positive nuclei in the OVLT largely restricted to the dorsal cap versus vascular core, respectively. However, in vitro patch-clamp recordings indicate 66% (23 of 35) of OVLT neurons were excited by bath application of both hypertonic NaCl and AngII. Similarly, in vivo single-unit recordings revealed that 52% (23 of 44) of OVLT neurons displayed an increased discharge to intracarotid injection of both hypertonic NaCl and AngII. In marked contrast to Fos immunoreactivity, neuroanatomical mapping of Neurobiotin-filled cells from both in vitro and in vivo recordings revealed that NaCl- and AngII-responsive neurons were distributed throughout the OVLT. Next, optogenetic excitation of OVLT neurons stimulated thirst but not salt appetite. Conversely, optogenetic inhibition of OVLT neurons attenuated thirst stimulated by hypernatremia or elevated AngII but not hypovolemia. Collectively, these findings provide the first identification of individual OVLT neurons that respond to both elevated NaCl and AngII concentrations to regulate thirst and body fluid homeostasis.SIGNIFICANCE STATEMENT Body fluid homeostasis requires the integration of neurohumoral signals to coordinate behavior, neuroendocrine function, and autonomic function. Extracellular NaCl concentrations and the peptide hormone angiotensin II (AngII) are two major neurohumoral signals that regulate body fluid homeostasis. Herein, we present the first compelling evidence that individual neurons located in the organum vasculosum of the lamina terminalis detect both NaCl and AngII. Furthermore, optogenetic interrogations demonstrate that these neurons play a pivotal role in the regulation of thirst stimulated by NaCl and AngII. These novel observations lay the foundation for future investigations for how such inputs as well as others converge onto unique organum vasculosum of the lamina terminalis neurons to coordinate body fluid homeostasis and contribute to disorders of fluid balance.

Perindopril mitigates LPS-induced cardiopulmonary oxidative and inflammatory damage via inhibition of renin angiotensin system, inflammation and oxidative stress

Aim: Renin–angiotensin system (RAS) is thought to have a noticeable effect in the pathophysiological injury in multiple organs by inducing different cellular and molecular reactions. The objective of the current study is to investigate the possible protective effects of perindopril against lipopolysaccharide (LPS)-induced cardiopulmonary oxidative and inflammatory damage in rats. Methods: To achieve this goal, animals were randomly divided into six groups: normal group, LPS group (3 mg/kg, i.p., single dose), perindopril-LPS treated group (1 mg/kg/day, i.p.), perindopril-LPS treated group (2 mg/kg/day, i.p.), and two perindopril negative groups (perindopril 1 or 2 mg/kg/day, i.p.) alone for seven days. Lungs and hearts tissue angiotensin II (Ang-II), angiotensin-1-7 (Ang-1-7), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were assessed using ELISA. Nuclear factor kappa-B-p65 (NF-κB-p65) was assessed using real time PCR, while protein kinase B (Akt) was evaluated by Western blot analysis. Furthermore, oxidative stress biomarkers and myeloperoxidase (MPO) enzyme were evaluated spectrophotometrically. Tissues inducible and endothelial nitric oxide synthases (iNOS and eNOS) were assessed immunohistochemically. Histopathological study was carried out to confirm our results. Results: LPS-intoxicated rats significantly elevated Ang-II, NF-κB-p65, Akt, and iNOS levels, coupled with significant down-regulation of Ang-1-7 and eNOS levels and corrected the oxidative stress biomarkers. Perindopril administration significantly attenuated the disturbances induced by LPS in a dose-dependent manner. Conclusion: Perindopril mitigates LPS-induced heart and lung damage through modulation of RAS, iNOS/eNOS, Akt, and NF-κB-p65 signaling pathways.

Pregnancy upregulates angiotensin type 2 receptor expression and increases blood flow in uterine arteries of rats.

Normal pregnancy is associated with decreased uterine vascular contraction and increased blood flow even though angiotensin II (AngII) levels are increased. AngII not only activates the angiotensin type 1 receptor (AT1R) to mediate vasoconstriction but also angiotensin type 2 receptor (AT2R) to cause vasodilation. We hypothesized that upregulation of AT2R expression and function accounts for increased uterine artery blood flow during pregnancy. Virgin, pregnant (at different days of gestation) and post-partum Sprague-Dawley rats were used to determine uterine artery hemodynamics using micro ultrasound and plasma angiotensin II levels by ELISA. Isolated uterine arteries were examined for AT1R and AT2R expression and isometric contraction/relaxation. Plasma AngII levels were steady up to mid-pregnancy, increased as pregnancy advanced, reaching a peak in late pregnancy, and then restored to pre-pregnant levels after delivery. The pattern of increase in AngII levels mirrored a parallel increase in uterine blood flow. AT1R expression did not change, but AT2R expression increased during pregnancy correlating with uterine blood flow increase. Treatment with the AT2R antagonist PD123319 reduced uterine arterial blood flow. Vasoconstriction to angiotensin II was blunted in pregnant rats. Treatment with PD123319 caused greater enhancement of AngII contraction in pregnant than virgin rats. Ex vivo exposure of estradiol to uterine arterial rings dose dependently upregulated AT2R expression, that was inhibited by estrogen receptor antagonist. These results demonstrate that elevated AngII levels during gestation induce an increase in uterine blood flow via heightened AT2R-mediated signaling. Estrogens appear to directly upregulate uterine vascular AT2R independent of any endogenous factors.

Abstract 010: Renal Proximal Tubule-derived Angiotensin Converting Enzyme 2 (ACE2) in Blood Pressure Regulation

Angiotensin Converting Enzyme 2 (ACE2) is a carboxypeptidase that metabolizes angiotensin II (AngII) to Ang1-7, playing a critical role in blood pressure (BP) regulation. Previous studies from our lab demonstrated that loss of ACE2 resulted in enhanced susceptibility to AngII hypertension and this was associated with elevated renal AngII levels. ACE2 is highly expressed in kidney, particularly in proximal tubule epithelia, and kidney cross-transplant experiments performed by our group revealed a novel mechanism by which ACE2 may be functioning to regulate BP. However, the exact cellular sources and sites of action where ACE2 regulates BP remain unclear. The objective of this study is to assess proximal tubule-derived ACE2 in BP regulation by studying a new mouse model generated in our laboratory: proximal tubule-specific ACE2 knock-out mice (referred to as ACE2 PTKO mice). ACE2 PTKO mice were generated by crossing the Ace2 -flox/flox conditional mouse line with the phosphoenolpyruvate carboxykinase (PEPCK)-Cre mouse (V. Haase, Vanderbilt). ACE2 PTKO mice and their littermate control mice were inbred 129/SvEv, co-caged, and only males were studied. ACE2 PTKO mice exhibited reduced ACE2 mRNA and protein in renal cortex compared to controls ( Ace2 mRNA relative expression: 0.47 ± 0.1 vs. 1.08 ± 0.1, N=3 vs 3, P &lt;0.05; relative ACE2 protein expression: 0.38 ± 0.04 vs. 1.15 ± 0.12, N=3 vs 3, P &lt;0.01). Measured with radiotelemetry, there were no differences in BPs between ACE2 PTKO mice and control mice (119 ± 1 vs 117 ± 2 mmHg, N=16 vs 16, p=NS) at baseline. However, ACE2 PTKO mice exhibited an enhanced hypertensive response during the first week of AngII infusion compared to control mice (139.9 ± 4.8 vs 152.8 ± 3.7 mmHg at day 7, N=16 vs 16, P =0.038). Furthermore, the ACE2 KO kidneys accumulated significantly higher renal AngII than control kidneys, measured at day4 (897±104 vs 536±115 fmol/g, P &lt;0.05). Our data demonstrate that proximal tubule-derived ACE2 plays a critical role in BP regulation during AngII infusion. The enhanced hypertensive response in ACE2KO mice is consistent with their intra-renal AngII levels, suggesting that that proximal tubule-derived ACE2 regulates BP via metabolizing AngII within the kidney.

Increased mitochondrial superoxide in the brain, but not periphery, sensitizes mice to angiotensin II-mediated hypertension

Angiotensin II (AngII) elicits the production of superoxide (O2•−) from mitochondria in numerous cell types within peripheral organs and in the brain suggesting a role for mitochondrial-produced O2•− in the pathogenesis of hypertension. However, it remains unclear if mitochondrial O2•− is causal in the development of AngII-induced hypertension, or if mitochondrial O2•− in the absence of elevated AngII is sufficient to increase blood pressure. Further, the tissue specific (i.e. central versus peripheral) redox regulation of AngII hypertension remains elusive. Herein, we hypothesized that increased mitochondrial O2•− in the absence of pro-hypertensive stimuli, such as AngII, elevates baseline systemic mean arterial pressure (MAP), and that AngII-mediated hypertension is exacerbated in animals with increased mitochondrial O2•− levels. To address this hypothesis, we generated novel inducible knock-down mouse models of manganese superoxide dismutase (MnSOD), the O2•− scavenging antioxidant enzyme specifically localized to mitochondria, targeted to either the brain subfornical organ (SFO) or peripheral tissues. Contrary to our hypothesis, knock-down of MnSOD either in the SFO or in peripheral tissues was not sufficient to alter baseline systemic MAP. Interestingly, when mice were challenged with chronic, peripheral infusion of AngII, only the MnSOD knock-down confined to the SFO, and not the periphery, demonstrated an increased sensitization and potentiated hypertension. In complementary experiments, over-expressing MnSOD in the SFO significantly decreased blood pressure in response to chronic AngII. Overall, these studies indicate that mitochondrial O2•− in the brain SFO works in concert with other AngII-dependent factors to drive an increase in MAP, as elevated mitochondrial O2•− alone, either in the SFO or peripheral tissues, failed to raise baseline blood pressure.

Abstract P247: Tumor Necrosis Factor-alpha Receptors (Type 1 &amp; Type 2) are Differentially Expressed in Renal Tissues During Chronic Dietary Intake of High Salt and Angiotensin II Treatment

Tumor necrosis factor-alpha (TNF-α) exerts natriuresis that is mediated by its’ receptor type 1 (TNFR1) while its’ receptor type 2 (TNFR2) is involved in mediating inflammatory renal injury. To determine the differential roles of these receptors in angiotensin II (AngII) induced salt-sensitive hypertension and associated renal injury, protein expressions of TNFR1 and TNFR2 were examined in mice (n=6-7 in each group) chronically treated with or without AngII (25 ng/min; implanted minipump) for 4 weeks which were fed either normal (NS; 0.4% NaCl) or high salt (HS; 4% NaCl) diets. Systemic blood pressure (SBP) in these mice was measured by tail-cuff plethysmography and 24 hour urine collections were made using metabolic cages at the start and at the end of treatment period when the kidneys were harvested after sacrificing the mice with euthanasia. Immuno-histochemical analysis of TNFR1 and TNFR2 proteins in renal slices was performed by measuring the staining area as well as the intensity of receptors’ immunoreactivities using NIS Elements Software (Nikon), which allowed the semi-quantitation of positive staining and the intensity of these proteins. The results were expressed in percent area of positive staining and the relative intensity. HS intake alone did not alter mean SBP (HS; 77±1 vs NS; 76±3 vs mmHg) but it caused an exaggeration of AngII induced increases in mean SBP (AngII+HS; 104±2 vs AngII+NS; 95±2 mmHg). The area of TNFR1 staining was higher in HS (6.0± 0.9 vs 3.2 ±0.7%; P&lt;0.05) than NS group but it was not significantly different between AngII+HS (5.0± 0.7%) and AngII+NS groups (6.3±0.7%). Similar qualitative differences were also observed in relative intensity in protein expressions. TNFR2 immunoreactivity was minimal in NS and HS groups but it was high in AngII+NS group and even greater in AngII + HS group. These data suggest that the increases in TNFR1 activity due to HS alone facilitate salt excretion that results no change in SBP in response to HS intake. However, such HS induced increases in TNFR1 activity was compromised in elevated AngII condition causing more salt retention and thus, exaggerated hypertensive response. On the other hand, HS induced increases in TNFR2 activity in elevated AngII condition facilitates enhanced renal injury response.

Xanthine oxidase inhibition alleviates the cardiac complications of insulin resistance: effect on low grade inflammation and the angiotensin system.

BackgroundWe have previously shown that hyperuricemia plays an important role in the vascular complications of insulin resistance (IR). Here we investigated the effect of xanthine oxidase (XO) inhibition on the cardiac complications of IR.MethodsIR was induced in rats by a high fructose high fat diet for 12 weeks. Allopurinol, a standard XO inhibitor, was administered in the last 4 weeks before cardiac hemodynamics and electrocardiography, serum glucose, insulin, tumor necrosis factor alpha (TNFα), 8-isoprostane, uric acid, lactate dehydrogenase (LDH) and XO activity were measured. Expression of cardiac angiotensin II (AngII) and angiotensin receptor 1 (AT1) were assessed by immunofluorescence.ResultsIR animals had significant hyperuricemia which was inhibited by allopurinol administration. IR was associated with impaired ventricular relaxation (reflected by a decreased diastolic pressure increment and prolonged diastolic duration) and XO inhibition greatly attenuated impaired relaxation. IR was accompanied by cardiac ischemia (reflected by increased QTc and T peak trend intervals) while XO inhibition alleviated the ECG abnormalities. When subjected to isoproterenol-induced ischemia, IR hearts were less resistant (reflected by larger ST height depression and higher LDH level) while XO inhibition alleviated the accompanying ischemia. In addition, XO inhibition prevented the elevation of serum 8-isoprostane and TNFα, and blocked elevated AngII and AT1 receptor expression in the heart tissue of IR animals. However, XO inhibition did not affect the developed hyperinsulinemia or dyslipidemia.ConclusionsXO inhibition alleviates cardiac ischemia and impaired relaxation in IR through the inhibition of low grade inflammation and the angiotensin system.

Angiotensin II mediates angiotensin converting enzyme type 2 internalization and degradation through an angiotensin II type I receptor-dependent mechanism.

Angiotensin-converting enzyme type 2 (ACE2) is a pivotal component of the renin-angiotensin system, promoting the conversion of angiotensin II (Ang-II) to Ang-(1-7). We previously reported that decreased ACE2 expression and activity contributes to the development of Ang-II-mediated hypertension in mice. The present study aimed to investigate the mechanisms involved in ACE2 downregulation during neurogenic hypertension. In ACE2-transfected Neuro-2A cells, Ang-II treatment resulted in a significant attenuation of ACE2 enzymatic activity. Examination of the subcellular localization of ACE2 revealed that Ang-II treatment leads to ACE2 internalization and degradation into lysosomes. These effects were prevented by both the Ang-II type 1 receptor (AT1R) blocker losartan and the lysosomal inhibitor leupeptin. In contrast, in HEK293T cells, which lack endogenous AT1R, Ang-II failed to promote ACE2 internalization. Moreover, this effect could be induced after AT1R transfection. Furthermore, coimmunoprecipitation experiments demonstrated that AT1R and ACE2 form complexes, and these interactions were decreased by Ang-II treatment, which also enhanced ACE2 ubiquitination. In contrast, ACE2 activity was not changed by transfection of AT2 or Mas receptors. In vivo, Ang-II-mediated hypertension was blunted by chronic infusion of leupeptin in wildtype C57Bl/6, but not in ACE2 knockout mice. Overall, this is the first demonstration that elevated Ang-II levels reduce ACE2 expression and activity by stimulation of lysosomal degradation through an AT1R-dependent mechanism.

Renal responses to intra‐arterial infusion of a peroxynitrite scavenging agent with or without angiotensin II in anesthetized rats (1134.4)

Peroxynitrite (OONO‐) is continually formed in the biological tissue by the spontaneous reaction between nitric oxide and superoxide. However, its potential role in the regulation of renal hemodynamics and excretory function in control condition as well as in the condition of elevated angiotensin II (AngII) level is not yet clearly defined. In the present study, we examined the responses to a OONO‐ scavenging agent, mercaptoethyl guanidine (MEG; 5 µg/kg/min for 75 min) infused directly into the left renal artery in anesthetized (Inactin; 100 mg/kg; i.p.) rats pretreated with (n=5) or without (n=5) AngII (1 ng/kg/min). The MEG dose was effective in blocking the increased renal blood flow (RBF) response to a bolus dose infusion of OONO‐ (40 µg/kg). RBF was recorded by the Transonic flow probe placed on the left renal artery and glomerular filtration rate (GFR) was determined by inulin clearance. In control rats (n=5), MEG infusion did not alter renal vascular resistance (RVR), RBF or GFR but caused significant decreases in urine flow (V, 9.5±1.4 to 7.8±1.5 μL/min/g; ‐20.2±6.7%), sodium excretion (UNaV, 1.69±0.38 to 1.34±0.43 μmol/min/g; ‐29.3±14.2%) and in fractional excretion of sodium (FENa, 1.04±0.33 to 0.87±0.36%; ‐29.3±15%). In a separate group (n=5), AngII treatment caused an increase of 18.0±4.1% in RVR (baseline value, b; 14.8±1.9 mmHg/mL/min/g) and the decreases of 14.4±3.6% in RBF (b, 7.2 mL/min/g), 17.1±6.2% in V (b, 6.7±2.3 μL/min/g), 35.6±14.4% in UNaV (b, 1.17±0.61 μmol/min/g) and 37.4±12.5% in FENa (b, 0.9±0.6%) without appreciable change in GFR. However, MEG infusion in these AngII treated rats caused similar decreases in renal excretory parameters (‐19.7±10.9% in V, ‐31.8±13.4% in UNaV and ‐33.3±8.4% in FENa) without significant changes in RVR, RBF or GFR. These data indicate that the endogenous OONO‐ exerts minimal hemodynamic effect but contributes to the maintenance of renal excretory function both in control condition as well as in the condition of elevated AngII level.

Ageing-related cardiomyocyte functional decline is sex and angiotensin II dependent

Clinically, heart failure is an age-dependent pathological phenomenon and displays sex-specific characteristics. The renin-angiotensin system mediates cardiac pathology in heart failure. This study investigated the sexually dimorphic functional effects of ageing combined with angiotensin II (AngII) on cardiac muscle cell function, twitch and Ca(2+)-handling characteristics of isolated cardiomyocytes from young (~13 weeks) and aged (~87 weeks) adult wild type (WT) and AngII-transgenic (TG) mice. We hypothesised that AngII-induced contractile impairment would be exacerbated in aged female cardiomyocytes and linked to Ca(2+)-handling disturbances. AngII-induced cardiomyocyte hypertrophy was evident in young adult mice of both sexes and accentuated by age (aged adult ~21-23 % increases in cell length relative to WT). In female AngII-TG mice, ageing was associated with suppressed cardiomyocyte contractility (% shortening, maximum rate of shortening, maximum rate of relaxation). This was associated with delayed cytosolic Ca(2+) removal during twitch relaxation (Tau ~20 % increase relative to young adult female WT), and myofilament responsiveness to Ca(2+) was maintained. In contrast, aged AngII-TG male cardiomyocytes exhibited peak shortening equivalent to young TG; yet, myofilament Ca(2+) responsiveness was profoundly reduced with ageing. Increased pro-arrhythmogenic spontaneous activity was evident with age and cardiac AngII overexpression in male mice (42-55 % of myocytes) but relatively suppressed in female aged transgenic mice. Female myocytes with elevated AngII appear more susceptible to an age-related contractile deficit, whereas male AngII-TG myocytes preserve contractile function with age but exhibit desensitisation of myofilaments to Ca(2+) and a heightened vulnerability to arrhythmic activity. These findings support the contention that sex-specific therapies are required for the treatment of age-progressive heart failure.

Angiotensin II Modulates Salty and Sweet Taste Sensitivities

Understanding the mechanisms underlying gustatory detection of dietary sodium is important for the prevention and treatment of hypertension. Here, we show that Angiotensin II (AngII), a major mediator of body fluid and sodium homeostasis, modulates salty and sweet taste sensitivities, and that this modulation critically influences ingestive behaviors in mice. Gustatory nerve recording demonstrated that AngII suppressed amiloride-sensitive taste responses to NaCl. Surprisingly, AngII also enhanced nerve responses to sweeteners, but had no effect on responses to KCl, sour, bitter, or umami tastants. These effects of AngII on nerve responses were blocked by the angiotensin II type 1 receptor (AT1) antagonist CV11974. In behavioral tests, CV11974 treatment reduced the stimulated high licking rate to NaCl and sweeteners in water-restricted mice with elevated plasma AngII levels. In taste cells AT1 proteins were coexpressed with αENaC (epithelial sodium channel α-subunit, an amiloride-sensitive salt taste receptor) or T1r3 (a sweet taste receptor component). These results suggest that the taste organ is a peripheral target of AngII. The specific reduction of amiloride-sensitive salt taste sensitivity by AngII may contribute to increased sodium intake. Furthermore, AngII may contribute to increased energy intake by enhancing sweet responses. The linkage between salty and sweet preferences via AngII signaling may optimize sodium and calorie intakes.

Angiotensin Type 1a Receptors in the Paraventricular Nucleus of the Hypothalamus Protect against Diet-Induced Obesity

Obesity is associated with increased levels of angiotensin-II (Ang-II), which activates angiotensin type 1a receptors (AT1a) to influence cardiovascular function and energy homeostasis. To test the hypothesis that specific AT1a within the brain control these processes, we used the Cre/lox system to delete AT1a from the paraventricular nucleus of the hypothalamus (PVN) of mice. PVN AT1a deletion did not affect body mass or adiposity when mice were maintained on standard chow. However, maintenance on a high-fat diet revealed a gene by environment interaction whereby mice lacking AT1a in the PVN had increased food intake and decreased energy expenditure that augmented body mass and adiposity relative to controls. Despite this increased adiposity, PVN AT1a deletion reduced systolic blood pressure, suggesting that this receptor population mediates the positive correlation between adiposity and blood pressure. Gene expression studies revealed that PVN AT1a deletion decreased hypothalamic expression of corticotrophin-releasing hormone and oxytocin, neuropeptides known to control food intake and sympathetic nervous system activity. Whole-cell patch-clamp recordings confirmed that PVN AT1a deletion eliminates responsiveness of PVN parvocellular neurons to Ang-II, and suggest that Ang-II responsiveness is increased in obese wild-type mice. Central inflammation is associated with metabolic and cardiovascular disorders and PVN AT1a deletion reduced indices of hypothalamic inflammation. Collectively, these studies demonstrate that PVN AT1a regulate energy balance during environmental challenges that promote metabolic and cardiovascular pathologies. The implication is that the elevated Ang-II that accompanies obesity serves as a negative feedback signal that activates PVN neurons to alleviate weight gain.