C-peptide increases forearm blood flow in patients with type 1 diabetes via a nitric oxide-dependent mechanism

Proinsulin C-peptide has been shown to increase muscle blood flow in type 1 diabetic patients. The underlying mechanism is not fully understood. The aim of this study was to evaluate if the vasodilator effect of C-peptide is mediated by nitric oxide (NO). Eleven type 1 diabetic patients were studied two times and randomized to administration of intravenous and intra-arterial infusion of C-peptide or saline. Forearm blood flow (FBF) was measured by venous occlusion plethysmography during infusion of C-peptide or saline before, during, and after NO synthase (NOS) blockade. Endothelium-dependent and -independent vasodilatation was evaluated by administration of acetylcholine and sodium nitroprusside, respectively. FBF increased by 35% during intravenous C-peptide (P < 0.01) but not during saline infusion (-2%, not significant). NOS blockade resulted in a more pronounced reduction in FBF during intravenous C-peptide than during saline infusion (-41 vs. -26%, P < 0.05). Intra-arterial C-peptide failed to increase FBF during NOS blockade. However, when C-peptide was given after the recovery from NOS blockade, FBF rose by 30% (P < 0.001). The vasodilator effects of acetylcholine and nitroprusside were not influenced by C-peptide. It is concluded that the stimulatory effect of C-peptide on FBF in type 1 diabetic patients is mediated via the NO system and that C-peptide increases basal endothelial NO levels.     

Angiotensin-(1-7): an update

The renin-angiotensin system is a major physiological regulator of arterial pressure and hydro-electrolyte balance. Evidence has now been accumulated that in addition to angiotensin (Ang) II other Ang peptides [Ang III, Ang IV and Ang-(1-7)], formed in the limited proteolysis processing of angiotensinogen, are importantly involved in mediating several actions of the RAS. In this article we will review our knowledge of the biological actions of Ang-(1-7) with focus on the puzzling aspects of the mediation of its effects and the interaction Ang-(1-7)-kinins. In addition, we will attempt to summarize the evidence that Ang-(1-7) takes an important part of the mechanisms aimed to counteract the vasoconstrictor and proliferative effects of Ang II.     

Angiotensin-(1-7) through Mas receptor up-regulates neuronal norepinephrine transporter via Akt and Erk1/2-dependent pathways

As angiotensin (Ang) (1-7) decreases norepinephrine (NE) content in the synaptic cleft, we investigated the effect of Ang-(1-7) on NE neuronal uptake in spontaneously hypertensive rats. [(3)H]-NE neuronal uptake was measured in isolated hypothalami. NE transporter (NET) expression was evaluated in hypothalamic neuronal cultures by western-blot. Ang-(1-7) lacked an acute effect on neuronal NE uptake. Conversely, Ang-(1-7) caused an increase in NET expression after 3 h incubation (40 ± 7%), which was blocked by the Mas receptor antagonist, a PI3-kinase inhibitor or a MEK1/2 inhibitor suggesting the involvement of Mas receptor and the PI3-kinase/Akt and MEK1/2-ERK1/2 pathways in the Ang-(1-7)-stimulated NET expression. Ang-(1-7) through Mas receptors stimulated Akt and ERK1/2 activities in spontaneously hypertensive rat neurons. Cycloheximide attenuated Ang-(1-7) stimulation of NET expression suggesting that Ang-(1-7) stimulates NET synthesis. In fact, Ang-(1-7) increased NET mRNA levels. Thus, we evaluated the long-term effect of Ang-(1-7) on neuronal NE uptake after 3 h incubation. Under this condition, Ang-(1-7) increased neuronal NE uptake by 60 ± 14% which was blocked by cycloheximide and the Mas receptor antagonist. Neuronal NE uptake and NET expression were decreased after 3 h incubation with an anti-Ang-(1-7) antibody. Ang-(1-7) induces a chronic stimulatory effect on NET expression. In this way, Ang-(1-7) may regulate a pre-synaptic mechanism in maintaining appropriate synaptic NE levels during hypertensive conditions.     

Adenoviral Delivery of Angiotensin-(1-7) or Angiotensin-(1-9) Inhibits Cardiomyocyte Hypertrophy via the Mas or Angiotensin Type 2 Receptor

The counter-regulatory axis of the renin angiotensin system peptide angiotensin-(1-7) [Ang-(1-7)] has been identified as a potential therapeutic target in cardiac remodelling, acting via the mas receptor. Furthermore, we recently reported that an alternative peptide, Ang-(1-9) also counteracts cardiac remodelling via the angiotensin type 2 receptor (AT₂R). Here, we have engineered adenoviral vectors expressing fusion proteins which release Ang-(1-7) [RAdAng-(1-7)] or Ang-(1-9) [RAdAng-(1-9)] and compared their effects on cardiomyocyte hypertrophy in rat H9c2 cardiomyocytes or primary adult rabbit cardiomyocytes, stimulated with angiotensin II, isoproterenol or arg-vasopressin. RAdAng-(1-7) and RAdAng-(1-9) efficiently transduced cardiomyocytes, expressed fusion proteins and secreted peptides, as demonstrated by western immunoblotting and conditioned media assays. Furthermore, secreted Ang-(1-7) and Ang-(1-9) inhibited cardiomyocyte hypertrophy (Control = 168.7±8.4 µm; AngII = 232.1±10.7 µm; AngII+RAdAng-(1-7) = 186±9.1 µm, RAdAng-(1-9) = 180.5±9 µm; P<0.05) and these effects were selectively reversed by inhibitors of their cognate receptors, the mas antagonist A779 for RAdAng-(1-7) and the AT₂R antagonist PD123,319 for RAdAng-(1-9). Thus gene transfer of Ang-(1-7) and Ang-(1-9) produces receptor-specific effects equivalent to those observed with addition of exogenous peptides. These data highlight that Ang-(1-7) and Ang-(1-9) can be expressed via gene transfer and inhibit cardiomyocyte hypertrophy via their respective receptors. This supports applications for this approach for sustained peptide delivery to study molecular effects and potential gene therapeutic actions.     

Angiotensin-(1-7) prevents diabetes-induced cardiovascular dysfunction

The aim of this study was to test the hypothesis that treatment with angiotensin-(1-7) [ANG-(1-7)] or ANG-(1-7) nonpeptide analog AVE-0991 can produce protection against diabetes-induced cardiovascular dysfunction. We examined the influence of chronic treatment (4 wk) with ANG-(1-7) (576 microg.kg(-1).day(-1) ip) or AVE-0991 (576 microg.kg(-1).day(-1) ip) on proteinuria, vascular responsiveness of isolated carotid and renal artery ring segments and mesenteric bed to vasoactive agonists, and cardiac recovery from ischemia-reperfusion in streptozotocin-treated rats (diabetes). Animals were killed 4 wk after induction of diabetes and/or treatment with ANG-(1-7) or AVE-0991. There was a significant increase in urine protein (231 +/- 2 mg/24 h) in diabetic animals compared with controls (88 +/- 6 mg/24 h). Treatment of diabetic animals with ANG-(1-7) or AVE-0991 resulted in a significant reduction in urine protein compared with vehicle-treated diabetic animals (183 +/- 16 and 149 +/- 15 mg/24 h, respectively). Treatment with ANG-(1-7) or AVE-0991 also prevented the diabetes-induced abnormal vascular responsiveness to norepinephrine, endothelin-1, angiotensin II, carbachol, and histamine in the perfused mesenteric bed and isolated carotid and renal arteries. In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischemia was significantly better in ANG-(1-7)- or AVE-0991-treated animals. These results suggest that activation of ANG-(1-7)-mediated signal transduction could be an important therapeutic strategy to reduce cardiovascular events in diabetic patients.     

Stabilization of Angiotensin-(1-7) in Cardioprotective Solutions

Angiotensin-(1-7) (Ang 1-7) has been previously studied in combination with an antioxidant containing preparation as a cardioprotective reperfusion solution. In this study a stability improvement of aqueous Ang 1-7 solutions was observed. However, no data was provided on the responsibilities and causes of the noticed stability enhancement. Therefore, the influence of pH and pharmaceutical additives as well as the effect of the single specific agents present in the antioxidant preparation such as α-ketoglutaric acid (α-KG), 5-hydroxymethylfurfural (5-HMF), N-acetyl-seleno-l-methionine (NASeLM) and N-acetyl-l-methionine (NALM) on the stability was evaluated. Analyses were performed by an HPLC method with fluorescence detection. Crucial instability was found in a pH range of 5.0–7.5 without addition of the antioxidative mixture. Zetasizing confirmed the presence of microparticles and MS studies showed no degradation products within 25 days. 5-HMF was identified as main component for stability enhancement of Ang 1-7 solution. By adding this substance the stability of the cardioprotective peptide solution can be prolonged and appears as a promising approach for transplant purposes.

     

The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism

Apelin is an endogenous ligand of the human orphan receptor APJ. We detected apelin-like immunoreactivity in the adipocytes, gastric mucosa, and Kupffer cells in the liver. We also detected apelin-like immunoreactivity localized within the endothelia of small arteries in various organs. Further, it was found that mean arterial pressure after the administration of apelin-12, apelin-13, and apelin-36 at a dose of 10 nmol/kg in anaesthetized rats was reduced by 26+/-5, 11+/-4, and 5+/-4 mm Hg, respectively. In the presence of a nitric oxide (NO) synthase inhibitor, the effect of apelin-12 on blood pressure was abolished. Furthermore, the administration of apelin-12 (10 nmol/kg) in rats produced a transitory elevation of the plasma nitrite/nitrate concentration from a basal level of 21.4+/-1.6 to 27.0+/-1.5 microM. Thus, apelin may lower blood pressure via a nitric oxide-dependent mechanism.     

Angiotensin-(1-7) stimulates oxidative stress in rat kidney

The effect of two different doses of angiotensin-(1-7) and angiotensin II on the oxidative stress generation was analyzed in rat kidney. Animals were injected intraperitoneally with a single dose of angiotensin-(1-7) or angiotensin II (20 or 50 nmol/kg body weight) and killed 3 h after injection. Production of thiobarbituric acid reactive substances (TBARS), measured as indicator of oxidative stress induction, was significantly increased in rat kidney after Ang-(1-7) administration up to 30% and 50% over controls, at 20 and 50 nmol/kg, respectively. Reduced glutathione (GSH), the most important soluble antioxidant defense in mammalian cells, showed a significant decrease of 13% and 20% at 20 and 50 nmol/kg of angiotensin-(1-7), respectively. When the antioxidant enzyme activities were determined, it was found that catalase activity was not altered by the assayed angiotensin-(1-7) doses while superoxide dismutase and glutathione peroxidase activities were significantly reduced by injection of 20 nmol/kg (34% and 13%, with respect to controls) and 50 nmol/kg of angiotensin-(1-7) (54% and 22%, respectively). In contrast, angiotensin II injections did not produce significant changes neither in TBARS levels nor in soluble and enzymatic defense parameters at the two doses used in this work. These results suggest that angiotensin-(1-7) is undoubtedly related to oxidative stress induction.     

Angiotensin-(1-7) abrogates mitogen-stimulated proliferation of cardiac fibroblasts

Previous studies showed that angiotensin-(1-7) [Ang-(1-7)] attenuates cardiac remodeling by reducing both interstitial and perivascular fibrosis. Although a high affinity binding site for Ang-(1-7) was identified on cardiac fibroblasts, the molecular mechanisms activated by the heptapeptide hormone were not identified. We isolated cardiac fibroblasts from neonatal rat hearts to investigate signaling pathways activated by Ang-(1-7) that participate in fibroblast proliferation. Ang-(1-7) reduced (3)H-thymidine, -leucine and -proline incorporation into cardiac fibroblasts stimulated with serum or the mitogen endothelin-1 (ET-1), demonstrating that the heptapeptide hormone decreases DNA, protein and collagen synthesis. The reduction in DNA synthesis by Ang-(1-7) was blocked by the AT((1-7)) receptor antagonist [d-Ala(7)]-Ang-(1-7), showing specificity of the response. Treatment of cardiac fibroblasts with Ang-(1-7) reduced the Ang II- or ET-1-stimulated increase in phospho-ERK1 and -ERK2. In contrast, Ang-(1-7) increased dual-specificity phosphatase DUSP1 immunoreactivity and mRNA, suggesting that the heptapeptide hormone increases DUSP1 to reduce MAP kinase phosphorylation and activity. Incubation of cardiac fibroblasts with ET-1 increased cyclooxygenase 2 (COX-2) and prostaglandin synthase (PGES) mRNAs, while Ang-(1-7) blocked the increase in both enzymes, suggesting that the heptapeptide hormone alters the concentration and the balance between the proliferative and anti-proliferative prostaglandins. Collectively, these results indicate that Ang-(1-7) participates in maintaining cardiac homeostasis by reducing proliferation and collagen production by cardiac fibroblasts in association with up-regulation of DUSP1 to reduce MAP kinase activities and attenuation of the synthesis of mitogenic prostaglandins. Increased Ang-(1-7) or agents that enhance production of the heptapeptide hormone may prevent abnormal fibrosis that occurs during cardiac pathologies.     

血管紧张素-(1-7)在动脉粥样硬化中的作用

血管紧张素-(1-7)［angiotensin(1-7), Ang-(1-7) ］是肾素-血管紧张素系 统(renin-angiotensin system, RAS)的新成员之一. 与血管紧张素Ⅱ(Ang Ⅱ)比 较,Ang-(1-7)在高血压及动脉粥样硬化等疾病中发挥着心血管保护作用. 最新研究 发现,Ang-(1-7)在平滑肌细胞增殖及迁移、内皮细胞功能、脂质代谢的这些动脉粥样硬化相关病理发展过程中起着重要作用,能够抑制动脉粥样硬化斑块的形成及加强斑块的稳定性. 本文就Ang-(1-7)与动脉粥样硬化疾病相关的最新研究进展进行综述,讨论Ang-(1-7)在动脉粥样硬化中的作用机制,也为Ang-(1-7)相关的ACE2/Ang -(1-7)/Mas轴作为未来临床治疗途径提供思路.     

Angiotensin-(1-7) attenuates hypertension in exercise-trained renal hypertensive rats

Angiotensin-(1-7) [ANG-(1-7)] plays a counterregulatory role to angiotensin II in the renin-angiotensin system. In trained spontaneous hypertensive rats, Mas expression and protein are upregulated in ventricular tissue. Therefore, we examined the role of ANG-(1-7) on cardiac hemodynamics, cardiac functions, and cardiac remodeling in trained two-kidney one-clip hypertensive (2K1C) rats. For this purpose, rats were divided into sedentary and trained groups. Each group consists of sham and 2K1C rats with and without ANG-(1-7) infusion. Swimming training was performed for 1 h/day, 5 days/wk for 4 wk following 1 wk of swimming training for acclimatization. 2K1C rats showed moderate hypertension and left ventricular hypertrophy without changing left ventricular function. Chronic infusion of ANG-(1-7) attenuated hypertension and cardiac hypertrophy only in trained 2K1C rats but not in sedentary 2K1C rats. Chronic ANG-(1-7) treatment significantly attenuated increases in myocyte diameter and cardiac fibrosis induced by hypertension in only trained 2K1C rats. The Mas receptor, ANG II type 2 receptor protein, and endothelial nitric oxide synthase phosphorylation in ventricles were upregulated in trained 2K1C rats. In conclusion, chronic infusion of ANG-(1-7) attenuates hypertension in trained 2K1C rats.     

Lack of Renoprotective Effect of Chronic Intravenous Angiotensin-(1-7) or Angiotensin-(2-10) in a Rat Model of Focal Segmental Glomerulosclerosis

Unopposed angiotensin (Ang) II-mediated cellular effects may lead to progressive glomerulosclerosis. While Ang-II can be locally generated in the kidneys, we previously showed that glomerular podocytes primarily convert Ang-I, the precursor of Ang-II, to Ang-(1-7) and Ang-(2-10), peptides that have been independently implicated in biological actions opposing those of Ang-II. Therefore, we hypothesized that Ang-(1-7) and Ang-(2-10) could be renoprotective in the fawn-hooded hypertensive rat, a model of focal segmental glomerulosclerosis. We evaluated the ability of 8–12 week-long intravenous administration of either Ang-(1-7) or Ang-(2-10) (100–400 ng/kg/min) to reduce glomerular injury in uni-nephrectomized fawn-hooded hypertensive rats, early or late in the disease. Vehicle-treated rats developed hypertension and lesions of focal segmental glomerulosclerosis. No reduction in glomerular damage was observed, as measured by either 24-hour urinary protein excretion or histological examination of glomerulosclerosis, upon Ang-(1-7) or Ang-(2-10) administration, regardless of peptide dose or disease stage. On the contrary, when given at 400 ng/kg/min, both peptides induced a further increase in systolic blood pressure. Content of Ang peptides was measured by parallel reaction monitoring in kidneys harvested at sacrifice. Exogenous administration of Ang-(1-7) and Ang-(2-10) did not lead to a significant increase in their corresponding intrarenal levels. However, the relative abundance of Ang-(1-7) with respect to Ang-II was increased in kidney homogenates of Ang-(1-7)-treated rats. We conclude that chronic intravenous administration of Ang-(1-7) or Ang-(2-10) does not ameliorate glomerular damage in a rat model of focal segmental glomerulosclerosis and may induce a further rise in blood pressure, potentially aggravating glomerular injury.     

Angiotensin-(1-7) attenuates diabetic nephropathy in Zucker diabetic fatty rats

Angiotensin (ANG)-(1-7) is known to attenuate diabetic nephropathy; however, its role in the modulation of renal inflammation and oxidative stress in type 2 diabetes is poorly understood. Thus in the present study we evaluated the renal effects of a chronic ANG-(1-7) treatment in Zucker diabetic fatty rats (ZDF), an animal model of type 2 diabetes and nephropathy. Sixteen-week-old male ZDF and their respective controls [lean Zucker rats (LZR)] were used for this study. The protocol involved three groups: 1) LZR + saline, 2) ZDF + saline, and 3) ZDF + ANG-(1-7). For 2 wk, animals were implanted with subcutaneous osmotic pumps that delivered either saline or ANG-(1-7) (100 ng·kg(-1)·min(-1)) (n = 4). Renal fibrosis and tissue parameters of oxidative stress were determined. Also, renal levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), ED-1, hypoxia-inducible factor-1α (HIF-1α), and neutrophil gelatinase-associated lipocalin (NGAL) were determined by immunohistochemistry and immunoblotting. ANG-(1-7) induced a reduction in triglyceridemia, proteinuria, and systolic blood pressure (SBP) together with a restoration of creatinine clearance in ZDF. Additionally, ANG-(1-7) reduced renal fibrosis, decreased thiobarbituric acid-reactive substances, and restored the activity of both renal superoxide dismutase and catalase in ZDF. This attenuation of renal oxidative stress proceeded with decreased renal immunostaining of IL-6, TNF-α, ED-1, HIF-1α, and NGAL to values similar to those displayed by LZR. Angiotensin-converting enzyme type 2 (ACE2) and ANG II levels remained unchanged after treatment with ANG-(1-7). Chronic ANG-(1-7) treatment exerts a renoprotective effect in ZDF associated with a reduction of SBP, oxidative stress, and inflammatory markers. Thus ANG-(1-7) emerges as a novel target for treatment of diabetic nephropathy.     

Inorganic nitrite attenuates NADPH oxidase-derived superoxide generation in activated macrophages via a nitric oxide-dependent mechanism

Oxidative stress contributes to the pathogenesis of many disorders, including diabetes and cardiovascular disease. Immune cells are major sources of superoxide (O₂^∙−) as part of the innate host defense system, but exaggerated and sustained O₂^∙− generation may lead to progressive inflammation and organ injuries. Previous studies have proven organ-protective effects of inorganic nitrite, a precursor of nitric oxide (NO), in conditions manifested by oxidative stress and inflammation. However, the mechanisms are still not clear. This study aimed at investigating the potential role of nitrite in modulating NADPH oxidase (NOX) activity in immune cells. Mice peritoneal macrophages or human monocytes were activated by lipopolysaccharide (LPS), with or without coincubation with nitrite. O₂^∙− and peroxynitrite (ONOO⁻) formation were detected by lucigenin-based chemiluminescence and fluorescence techniques, respectively. The intracellular NO production was measured by DAF-FM DA fluorescence. NOX isoforms and inducible NO synthase (iNOS) expression were detected by qPCR. LPS increased both O₂^∙− and ONOO⁻ production in macrophages, which was significantly reduced by nitrite (10 µmol/L). Mechanistically, the effects of nitrite are (1) linked to increased NO generation, (2) similar to that observed with the NO donor DETA-NONOate, and (3) can be abolished by the NO scavenger carboxy-PTIO or by the xanthine oxidase (XO) inhibitor febuxostat. Nox2 expression was increased in activated macrophages, but was not influenced by nitrite. However, nitrite attenuated LPS-induced upregulation of iNOS expression. Similar to that observed in mice macrophages, nitrite also reduced O₂^∙− generation in LPS-activated human monocytes. In conclusion, XO-mediated reduction of nitrite attenuates NOX activity in activated macrophages, which may modulate the inflammatory response.     

Angiotensin-(1--7) in the ovine fetus

In the adult animal, ANG-(1-7) may counterbalance some effects of ANG II. Its effects in the fetus are unknown. Basal ANG-(1-7), ANG I, ANG II, and renin concentrations were measured in plasma from ovine fetuses and their mothers (n = 10) at 111 days of gestation. In the fetus, concentrations of ANG I, ANG-(1-7), and ANG II were 86 +/- 21, 13 +/- 2, and 14 +/- 2 fmol/ml, respectively. In the ewe, concentrations of ANG I were significantly lower (20 +/- 4 fmol/ml, P < 0.05) as were concentrations of ANG-(1-7) (2.9 +/- 0.6 fmol/ml), whereas ANG II concentrations were not different (10 +/- 1 fmol/ml). Plasma renin concentrations were higher in the fetus (4.8 +/- 1.1 pmol ANG I x ml(-1) x h(-1)) than in the ewe (0.9 +/- 0.2 pmol x ml(-1) x h(-1), P < 0.05). Infusion of ANG-(1-7) (approximately 9 microg/h) for a 3-day period caused a significant increase in plasma concentrations of ANG-(1-7) reaching a maximum of 448 +/- 146 fmol/ml on day 3 of infusion. Plasma levels of ANG I and II as well as renin were unchanged by the infusion. Urine flow rate, glomerular filtration rate, and fetal arterial blood pressure did not change and were not different than values in fetuses receiving a saline infusion for 3 days (n = 5). However, the osmolality of amniotic and allantoic fluid was significantly higher in fetuses that received ANG-(1-7). Also, compared with the saline-infused animals, mRNA expression levels of renin, the AT(1) receptor, and AT(2) receptor were elevated in kidneys of fetuses that received infusions of ANG-(1-7). Infusion of an ANG-(1-7) antagonist ([D-Ala(7)]-ANG-(1-7), 20 microg/h) for 3 days had no effect on fetal blood pressure or renal function. In conclusion, although infusion of ANG-(1-7) did not affect fetal urine flow rate, glomerular filtration rate, or blood pressure, changes in fetal fluids and gene expression indicate that ANG-(1-7) may play a role in the fetal kidney.     

Angiotensin-(1-7) suppresses the number and function of the circulating fibrocytes by upregulating endothelial nitric oxide synthase expression

There is growing evidence suggesting that circulating fibrocytes (CFs) play a pivotal role in tissue repair and fibrosis. In contrast, in recent studies, angiotensin-(1-7) [Ang-(1-7)] has been shown to antagonize fibrosis. The purpose of this study was to examine the direct effect of Ang-(1-7) on CFs. Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation. Using laser scanning confocal microscopy, CFs were identified as adherent cells that stained positive for both CD34 and collagen-I. After 14 days of culture, CFs were stimulated with Ang-(1-7) at concentrations of 10 nM, 100 nM, 1 μM or 10 μM, in the absence and presence of pretreatment with A-779, N(G)-nitro-L-arginine methyl ester (L-NAME) or both, for 24, 48 or 72 h. The number of cells, cellular proliferation, and level of apoptosis were determined by hematoxylin and eosin staining, the Cell Counting Kit-8 (CCK8) assay and the annexin V/propidium iodide binding assay, respectively. The collagen content of CFs was measured by the concentration of hydroxyproline, which was detected using the enzymatic digestion method. The expression of endothelial nitric oxide synthase (eNOS) was assayed by western Blot analysis, while nitric oxide (NO) generation was detected using the Griess method. We found that Ang-(1-7) increases apoptosis and eNOS/NO production in CFs. In addition, Ang-(1-7) decreases the number, proliferative capacity and collagen-secretion of CFs in a concentration- and time-dependent manner. These data suggest that Ang-(1-7) suppresses the both the number and function of CFs possibly by increasing eNOS/NO production in the CFs.     

Angiotensin-(1-7) Attenuates Kidney Injury Due to Obstructive Nephropathy in Rats

Background

Angiotensin-(1–7) [Ang-(1–7)] counteracts many actions of the renin-angiotensin-aldosterone system. Despite its renoprotective effects, extensive controversy exists regarding the role of Ang-(1–7) in obstructive nephropathy, which is characterized by renal tubulointerstitial fibrosis and apoptosis.

Methods

To examine the effects of Ang-(1–7) in unilateral ureteral obstruction (UUO), male Sprague-Dawley rats were divided into three groups: control, UUO, and Ang-(1–7)-treated UUO rats. Ang-(1–7) was continuously infused (24 μg/[kg·h]) using osmotic pumps. We also treated NRK-52E cells in vitro with Ang II (1 μM) in the presence or absence of Ang-(1–7) (1 μM), Mas receptor antagonist A779 (1 μM), and Mas receptor siRNA (50 nM) to examine the effects of Ang-(1–7) treatment on Ang II-stimulated renal injury via Mas receptor.

Results

Angiotensin II (Ang II) and angiotensin type 1 receptor (AT₁R) protein expression was higher in UUO kidneys than in controls. Ang-(1–7) treatment also decreased proapoptotic protein expression in UUO kidneys. Ang-(1–7) also significantly ameliorated TUNEL positive cells in UUO kidneys. Additionally, Ang-(1–7) reduced profibrotic protein expression and decreased the increased tumor growth factor (TGF)-β1/Smad signaling present in UUO kidneys. In NRK-52E cells, Ang II induced the expression of TGF-β1/Smad signaling effectors and proapoptotic and fibrotic proteins, as well as cell cycle arrest, which were attenuated by Ang-(1–7) pretreatment. However, treatment with A779 and Mas receptor siRNA enhanced Ang II-induced apoptosis and fibrosis. Moreover, Ang II increased tumor necrosis factor-α converting enzyme (TACE) and decreased angiotensin-converting enzyme 2 (ACE2) expression in NRK-52E cells, while pretreatment with Ang-(1–7) or A779 significantly inhibited or enhanced these effects, respectively.

Conclusion

Ang-(1–7) prevents obstructive nephropathy by suppressing renal apoptosis and fibrosis, possibly by regulating TGF-β1/Smad signaling and cell cycle arrest via suppression of AT₁R expression. In addition, Ang-(1–7) increased and decreased ACE2 and TACE expression, respectively, which could potentially mediate a positive feedback mechanism via the Mas receptor.     

Angiotensin-(1-7) attenuates high glucose-induced proximal tubular epithelial-to-mesenchymal transition via inhibiting ERK1/2 and p38 phosphorylation

AimsThe kidney is an important target for both Angiotensin II and angiotensin-(1–7) [Ang-(1–7)] in the renin–angiotensin system. However, the renal function of Ang-(1–7) remains unclear. This study is aimed at investigating the effect of Ang-(1–7) on high glucose-induced epithelial to mesenchymal transition (EMT) in cultured renal epithelial cells.Main methodsCultured renal epithelial (NRK-52E) cell line was used in the experiment. Fluorescence immunocytochemistry was performed to observe α-smooth muscle actin (α-SMA). Real-time PCR and Western blot were used to determine mRNA and protein levels. Enzyme-linked immunosorbent assay was used to measure the concentration of transforming growth factor-β1 (TGF-β1) in the culture media.Key findingsHigh glucose-induced decreased in both angiotensin-converting enzyme-related carboxypeptidase (ACE2) and Mas mRNA levels. Meanwhile, high glucose induced increases in α-SMA and vimentin, decreases in E-cadherin, elevations in TGF-β1 and fibronectin secretions. Ang-(1–7) partially reversed high glucose-induced changes in α-SMA, vimentin, E-cadherin, TGF-β1 and fibronectin. High glucose stimulated ERK, p38 and JNK phosphorylation and Ang-(1–7) reversed the changes in ERK and p38 but not JNK phosphorylation.SignificanceInhibition and insufficiency in ACE2-Ang-(1–7)–Mas axis under high glucose condition participate EMT. Supplementation of Ang-(1–7) attenuates high glucose-induced EMT. ERK and p38 intracellular signaling pathways, not JNK, mediate the effect of Ang-(1–7) on EMT.     

Transactivation of ErbB Family of Receptor Tyrosine Kinases Is Inhibited by Angiotensin-(1-7) via Its Mas Receptor

Transactivation of the epidermal growth factor receptor (EGFR or ErbB) family members, namely EGFR and ErbB2, appears important in the development of diabetes-induced vascular dysfunction. Angiotensin-(1–7) [Ang-(1–7)] can prevent the development of hyperglycemia-induced vascular complications partly through inhibiting EGFR transactivation. Here, we investigated whether Ang-(1–7) can inhibit transactivation of ErbB2 as well as other ErbB receptors in vivo and in vitro. Streptozotocin-induced diabetic rats were chronically treated with Ang-(1–7) or AG825, a selective ErbB2 inhibitor, for 4 weeks and mechanistic studies performed in the isolated mesenteric vasculature bed as well as in cultured vascular smooth muscle cells (VSMCs). Ang-(1–7) or AG825 treatment inhibited diabetes-induced phosphorylation of ErbB2 receptor at tyrosine residues Y1221/22, Y1248, Y877, as well as downstream signaling via ERK1/2, p38 MAPK, ROCK, eNOS and IkB-α in the mesenteric vascular bed. In VSMCs cultured in high glucose (25 mM), Ang-(1–7) inhibited src-dependent ErbB2 transactivation that was opposed by the selective Mas receptor antagonist, D-Pro⁷-Ang-(1–7). Ang-(1–7) via Mas receptor also inhibited both Angiotensin II- and noradrenaline/norephinephrine-induced transactivation of ErbB2 and/or EGFR receptors. Further, hyperglycemia-induced transactivation of ErbB3 and ErbB4 receptors could be attenuated by Ang-(1–7) that could be prevented by D-Pro⁷-Ang-(1–7) in VSMC. These data suggest that Ang-(1–7) via its Mas receptor acts as a pan-ErbB inhibitor and might represent a novel general mechanism by which Ang-(1–7) exerts its beneficial effects in many disease states including diabetes-induced vascular complications.