Up-regulation of AT(1) and AT(2) receptors in postinfarcted hypertrophied myocytes and stretch-mediated apoptotic cell death

To determine whether up-regulation of AT(1) and AT(2) receptors occurred in hypertrophied myocytes after infarction and whether AT(2) played a role in stretch-mediated apoptosis, left ventricular myocytes were dissociated from the surviving portion of the wall 8 days after coronary occlusion and cardiac failure in rats. Control cells were obtained from sham-operated animals. Myocytes were stretched in an equibiaxial stretch apparatus and angiotensin II (Ang II) formation and cell death were measured 3 and 12 hours later. AT(1) and AT(2) proteins were evaluated in freshly isolated myocytes and after stretch. The effects of AT(1) and AT(2) antagonists on stretch-induced Ang II synthesis and apoptosis were also established. Myocardial infarction increased AT(1) and AT(2) in myocytes and stretch further up-regulated these receptors. Ang II levels were higher in postinfarcted myocytes and this peptide increased with the duration of stretch in both groups of cells. Similarly, apoptosis increased with time in control and postinfarcted myocytes. Absolute values of Ang II and apoptosis were greater in myocytes from infarcted hearts at 3 and 12 hours after stretch. Addition of AT(1) blocker to cultures inhibited stretch-activated apoptosis in both myocyte populations as well as the generation of Ang II in postinfarcted myocytes. In contrast, AT(2) antagonists had no impact on these cellular events. In conclusion, Ang II stimulated cell death through AT(1) receptor activation, whereas ligand binding to AT(2) receptor did not alter Ang II concentration and apoptosis in normal and postinfarcted hypertrophied myocytes.     

Experimental depression induces renal oxidative stress in rats

Depression has been associated to inflammatory and oxidative events. Previous report has shown renal oxidative stress in patients with depression. In order to analyze if depressive status is related to renal oxidative and inflammatory events, Sprague Dawley rats were submitted to forced swimming test (FST) and the renal oxidative metabolism, monocyte-macrophage infiltration and Angiotensin II (Ang II) expression were determined. Rats were submitted to FST daily (30 min) for 15 days. Motor activity was analyzed before FST. Kidney sections were homogenized to measure nitric oxide (NO), malondialdehyde (MDA), reduced glutathione (GSH) and catalase activity by enzymatic and biochemical methods. Renal frozen sections were studied for superoxide anion (O₂-), monocyte/macrophage infiltration and Ang II expression by histochemical and immunofluorescence methods. In addition, three groups of FST rats were treated with losartan, sertraline or water for 18 days with further renal O₂-analysis. In the FST group, struggle time, motor activity, food intake and body weight gain were found decreased. Increased number of glomerular, interstitial and tubular O₂-positive cells was observed in FST rats. High renal content of nitrite/nitrate (NO), MDA and decreased amount of GSH were found in FST rats. Values of renal ED-1 or Ang II positive cells in FST rats remained similar to controls; however, AT1 receptor blocking (losartan) and sertraline reduced both depressive-like behavior and renal O₂-expression. These data suggests that depression-like behavior in rats is involved in kidney oxidative stress probably mediated by AT1 receptors.     

Angiotensin II increases connective tissue growth factor in the kidney

Connective tissue growth factor (CTGF) has been described as a novel fibrotic mediator. CTGF is overexpressed in several kidney diseases and is induced by different factors involved in renal injury. Angiotensin II (AngII) participates in the pathogenesis of kidney damage, contributing to fibrosis; however, whether AngII regulates CTGF in the kidney has not been explored. Systemic infusion of AngII into normal rats for 3 days increased renal CTGF mRNA and protein levels. At day 7, AngII-infused rats presented overexpression of CTGF in glomeruli, tubuli, and renal arteries, as well as tubular injury and elevated fibronectin deposition. Only treatment with an AT(1) receptor antagonist, but not an AT(2), diminished CTGF and fibronectin overexpression and ameliorated tubular damage. In rats with immune complex nephritis, renal overexpression of CTGF was diminished by the ACE inhibitor quinapril, correlated with a diminution in fibrosis. In cultured renal cells (mesangial and tubular epithelial cells) AngII, via AT(1), increased CTGF mRNA and protein production, and a CTGF antisense oligonucleotide decreased AngII-induced fibronectin synthesis. Our data show that AngII regulates CTGF in the kidney and cultured in mesangial and tubular cells. This novel finding suggests that CTGF could be a mediator of the profibrogenic effects of AngII in the kidney.     

AT1 receptor-mediated angiotensin II activation and chemotaxis of T lymphocytes

Angiotensin II (Ang II), a central renin-angiotensin system (RAS) effector molecule, and its receptors, AT(1) and AT(2), have been shown to be involved in the inflammatory aspects of different diseases, however the cellular mechanisms underlying the regulation of immunity are not fully understood. In this work, using spleen-derived CD4(+) and CD8(+) T lymphocytes activated in vitro, we tested the influence of Ang II on different aspects of the T cell function, such as activation and adhesion/transmigration through endothelial basal membrane proteins. The addition of 10(-8)M Ang II did not change any of the parameters evaluated. However, 10(-6)M losartan, an AT(1) receptor antagonist: (i) reduced the percentage of CD25(+) and CD69(+) cells of both subsets; (ii) inhibited adhesion of these cells to fibronectin or laminin by 53% or 76%, respectively and (iii) significantly reduced transmigration through fibronectin or laminin by 57% or 43%, respectively. In addition, 10(-6)M captopril, an angiotensin-converting enzyme inhibitor had similar effects to Ang II, however its effects were reverted by exogenous Ang II (10(-8)M). None of these responses was modified by 10(-7)M PD123319, an AT(2) antagonist. These data reinforce the notion of endogenous production of Ang II by T cells, which is important for T cell activation, and adhesion/transmigration induced on interaction with basal membrane proteins, possibly involving AT(1) receptor activation. Moreover, AT(1) receptor expression is 10-fold higher in activated T lymphocytes compared with naive cells, but AT(2) receptor expression did not change after T cell receptor triggering.     

Human renal fibroblasts derived from normal and fibrotic kidneys show differences in increase of extracellular matrix synthesis and cell proliferation upon angiotensin II exposure

Angiotensin II (Ang II) as a vasoactive hormone may be involved in progressive renal interstitial fibrosis. We investigated the influence of Ang II on cell proliferation and synthesis of extracellular matrix (collagen I, III and fibronectin) in human renal fibroblasts derived from normal (TK 173 cell line) and fibrotic (TK 188 cell line) kidneys which possess both Ang II type l and type 2 (AT1 and AT2) receptors. Incubation of the cells with Ang II increased the cell proliferation and the synthesis of extracellular matrix significantly in both cell lines. However, proliferation and extracellular matrix synthesis showed a greater increase in the cells derived from the fibrotic kidney. The Ang II mediated effect on cell proliferation and extracellular matrix synthesis was diminished in the presence of the AT1 receptor blocker losartan in both cell lines. No inhibition was observed using the AT2 receptor blocker PD123319. Ang II induced cell proliferation could be completely inhibited by incubation with human TGF-beta1 antibody. Incubation with Ang II did not affect TGF beta 1 production but in untreated cells TGF-beta 1 content was higher in the cells derived from the fibrotic kidney. This might be the reason for the more sensitive reaction on exposure to Ang II.     

Angiotensin II up-regulates angiotensin I-converting enzyme (ACE), but down-regulates ACE2 via the AT1-ERK/p38 MAP kinase pathway

The recent discovery of the angiotensin II (Ang II)-breakdown enzyme, angiotensin I converting enzyme (ACE) 2, suggests the importance of Ang II degradation in hypertension. The present study explored the signaling mechanism by which ACE2 is regulated under hypertensive conditions. Real-time PCR and immunohistochemistry showed that ACE2 mRNA and protein expression levels were high, whereas ACE expression levels were moderate in both normal kidney and heart. In contrast, patients with hypertension showed marked ACE up-regulation and ACE2 down-regulation in both hypertensive cardiopathy and, particularly, hypertensive nephropathy. The inhibition of ACE2 expression was shown to be associated with ACE up-regulation and activation of extracellular regulated (ERK)1/2 and p38 mitogen-activated protein (MAP) kinases. In vitro, Ang II was able to up-regulate ACE and down-regulate ACE2 in human kidney tubular cells, which were blocked by an angiotensin II (AT)1 receptor antagonist (losartan), but not by an AT2 receptor blocker (PD123319). Furthermore, blockade of ERK1/2 or p38 MAP kinases by either specific inhibitors or a dominant-negative adenovirus was able to abolish Ang II-induced ACE2 down-regulation in human kidney tubular cells. In conclusion, Ang II is able to up-regulate ACE and down-regulate ACE2 expression levels under hypertensive conditions both in vivo and in vitro. The AT1 receptor-mediated ERK/p38 MAP kinase signaling pathway may be a key mechanism by which Ang II down-regulates ACE2 expression, implicating an ACE/ACE2 imbalance in hypertensive cardiovascular and renal damage.     

Angiotensin II subtype 1A (AT1A) receptors in the rat sensory vagal complex: subcellular localization and association with endogenous angiotensin

Angiotensin II (Ang II) type 1 (AT1) receptors are prevalent in the sensory vagal complex including the nucleus tractus solitarii (NTS) and area postrema, each of which has been implicated in the central cardiovascular effects produced by Ang II. In rodents, these actions prominently involve the AT1A receptor. Thus, we examined the electron microscopic dual immunolabeling of antisera recognizing the AT1A receptor and Ang II to determine interactive sites in the sensory vagal complex of rat brain. In both the area postrema and adjacent dorsomedial NTS, many somatodendritic profiles were dually labeled for the AT1A receptor and Ang II. In these profiles, AT1A receptor-immunoreactivity was often seen in the cytoplasm beneath labeled portions of the plasma membrane and in endosome-like granules as well as Golgi lamellae and outer nuclear membranes. In addition, AT1A receptor labeling was detected on the plasma membrane and in association with cytoplasmic membranes in many small axons and axon terminals. These terminals were morphologically heterogeneous containing multiple types of vesicles and forming either inhibitory- or excitatory-type synapses. In the area postrema, AT1A receptor labeling also was detected in many non-neuronal cells including glia, capillary endothelial cells and perivascular fibroblasts that were less prevalent in the NTS.We conclude that in the rat sensory vagal complex, AT1A receptors are strategically positioned for involvement in modulation of the postsynaptic excitability and intracrine hormone-like effects of Ang II. In addition, these receptors have distributions consistent with diverse roles in regulation of transmitter release, regional blood flow and/or vascular permeability.     

Specific MAP-kinase blockade protects against renal damage in homozygous TGR(mRen2)27 rats

Angiotensin II (AngII) plays an important role in renal damage by acting on hemodynamics, cell-growth, proliferation, and fibrosis, mainly by effects on the AngII type 1 (AT(1)) receptor. The AT(1) receptor activates several intracellular signaling molecules such as mitogen-activated protein kinases extracellular signal-regulated kinase (ERK) and p38, but their role in AngII-mediated renal damage is not well characterized. We therefore investigated whether pharmacologic blockade of ERK and p38 could prevent renal damage in high-renin homozygous transgenic rats (Ren2), with the effects of an AT(1) receptor antagonist (AT(1)-RA) as a reference. Seven-week-old homozygous Ren2 rats were treated with low-dose AT(1)-RA candesartan, ERK inhibitor tyrphostin, or p38 inhibitor SB239063 for 4 weeks. Untreated Ren2 and SD rats served as controls. Blood pressure was measured at 7 and 11 weeks. At 11 weeks, plasma renin activity (PRA) and serum aldosterone were determined, and the animals were killed. Kidney sections were scored for glomerular and interstitial smooth muscle actin and glomerular desmin expression as early markers for renal damage. Mesangial matrix expansion was determined as a marker for structural damage. PRA and aldosterone levels were elevated in untreated Ren2 rats in comparison to SD controls. AT(1)-RA further increased PRA but decreased aldosterone. All parameters of renal damage were elevated in untreated Ren2 rats. Blood pressure was not elevated at week 7 in Ren2 and not affected by either treatment. Mild signs of hypertensive damage were found in untreated Ren2 rats. All interventions significantly diminished damage to glomerular epithelium and interstitium. In addition, AT(1) receptor and p38 blockade reduced mesangial matrix expansion. In homozygous Ren2 rats, renal damage was ameliorated by a nonhypotensive dose of an AT(1)-RA and, similarly, by blockade of ERK or p38. This suggests that ERK and p38 are involved in AngII-mediated renal damage.     

胚胎分化/发育基因pax2与WT1的序列启动在肾小管上皮细胞逆向分化中的意义

目的： 通过观察肾小管上皮细胞(TEC)胚胎分化/发育关键基因Wilm’s肿瘤基因（WT1）和pax2的序列启动，探讨TEC逆向分化的分子机制及病理生理学意义。方法：从胚胎肾、肾脏损伤模型及体外细胞培养3方面入手，通过RT-PCR、免疫组织化学等技术，研究TEC逆向分化时，胚胎期控制TEC分化/发育的关键基因pax2和WT1的表达、pax2和WT1之间的关系以及它们与TEC逆向分化间的关系。结果：（1）胚胎期：pax2和WT1 mRNA先后在胚胎11.5 d及14 d时开始出现表达并逐渐增强，出生14 d后仅有痕量表达。免疫组化显示，成年大鼠中仅肾小球足突细胞表达WT1，TEC中pax2和WT1表达阴性。（2）5/6慢性肾损伤模型：损伤第2周，部分TEC重新出现pax2和WT1的表达，第4周达高峰，两者表达趋势相吻合。pax2在第10周尚有一个新的表达高峰，随后逐渐下降至痕量。（3）TEC体外培养：经炎性因子IL-1α(10 μg/L)、AngII（10-9 mol/L）掺入培养后，TEC分别在0.5、24 h内重新表达pax2 和WT1，随后α-SMA出现高表达，细胞呈现间充质样细胞特征。以WT1中和抗体、AngII 受体阻断剂封闭WT1和pax2基因的作用后，α-SMA表达水平明显降低，细胞基本保持原有特征不变。结论：控制胚胎肾分化/发育的关键基因pax2和WT1在成年肾遭受损伤时，可获得重新表达，呈现序列启动现象，TEC同时出现胚胎间充质细胞特征；pax2和WT1的重新表达与细胞浸浴环境（高浓度细胞因子）密切相关，可能作为内在启动机制参与TEC的逆向分化。     

Role of angiotensin II and angiotensin type-1 receptor in scorpion venom-induced cardiac and aortic tissue inflammation

Scorpion stings are mainly associated with cardiovascular disturbances that may be the cause of death. In this study, the involvement of angiotensin II (Ang II) in cardiac and aortic inflammatory response was studied. Mice were injected with Androctonus australis hector (Aah) scorpion venom (0.5 mg/kg, subcutaneously), in the presence or absence of an angiotensin converting enzyme (ACE) inhibitor, captopril (15 mg/kg/day/1 day intraperitoneally) or an angiotensin type-1 receptor (AT1R) antagonist, valsartan (15 mg/kg/day/15 days, orally). In the envenomed group, results revealed severe tissue alterations with a concomitant increase of metabolic enzymes (CK and CK-MB) in sera. An important inflammatory cell (neutrophil and eosinophil) infiltration into the heart and aorta were observed, accompanied by imbalanced redox status (NO, MDA, catalase and GSH) and high cytokine levels (IL-6 and TNF-α) in sera with the expression of MMP-2 and MMP-9 metalloproteinases. However, the blockade of the actions of AngII by the ACE inhibitor or by the AT1R antagonist prevented cardiac and aortic tissue alterations, inflammatory cell infiltration, as well as the oxidative stress generation and cytokine and metalloproteinase expression. These results suggest the involvement of AngII, through its AT1R in the inflammation induced by Aah venom, in the heart and the aorta.     

Angiotensin II type 1 and type 2 receptors play opposite roles in regulating the barrier function of kidney glomerular capillary wall

Although angiotensin II (Ang II) type 1 receptor antagonist ameliorates proteinuria, its pharmacological mechanism and the differential roles of Ang II type 1 receptor (AT1R) and type 2 receptor (AT2R) are not well understood. We analyzed the effect of Ang II type 1 receptor antagonist on proteinuria caused by antibody against nephrin, a functional molecule of glomerular slit diaphragm and dysfunction of which is involved in the development of proteinuria in several glomerular diseases. We show here that AT1R antagonist ameliorated proteinuria by preventing a reduction in the functional molecules of the slit diaphragm. We also analyzed the role of AT1R- or AT2R-mediated actions on the expression of the slit diaphragm molecules in an in vivo study of normal rat and in an in vitro study of cultured podocytes. AT1R-mediated action hampered the mRNA expression of the slit diaphragm molecules, whereas AT2R-mediated action enhanced it. These findings indicate that Ang II receptor subtypes play opposite roles in regulating the barrier function of glomerular capillary wall and that the enhancement of AT2R stimulation may serve as a potential therapeutic strategy for proteinuria.     

Angiotensin AT1 receptors in paraventricular nucleus contribute to sympathetic activation and enhanced cardiac sympathetic afferent reflex in renovascular hypertensive rats

Sympathetic activity is enhanced in hypertension, which contributes to the pathogenesis of hypertension and progression of organ damage. The cardiac sympathetic afferent reflex (CSAR) is enhanced in renovascular hypertension and involved in the sympathetic activation. The present study was designed to investigate whether angiotensin II (Ang II) and Ang II type 1 (AT(1)) receptors in paraventricular nucleus (PVN) contribute to the enhanced CSAR and sympathetic outflow in experimental renovascular hypertensive rats. Hypertension was induced by the two-kidney one-clip (2K1C) method. The normotensive rats underwent sham operation (Sham). Acute experimentation was carried out at the end of the 4th week. Under urethane and α-chloralose anaesthesia, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) were recorded in rats with sino-aortic denervation and cervical vagotomy. The AT(1) receptor expression was determined with Western blot. The CSAR was evaluated by the response of RSNA and MAP to epicardial application of 1.0 nmol of capsaicin. The AT(1) receptor expression in the PVN was increased, and Ang II in the PVN augmented the enhanced CSAR and RSNA in 2K1C rats. The effects of Ang II were abolished by pretreatment with the AT(1) receptor antagonist, losartan, in 2K1C rats. Losartan in the PVN normalized the enhanced CSAR and decreased the RSNA and MAP in 2K1C rats. These results indicate that the increased activity of AT(1) receptors in the PVN contributes to the enhanced CSAR and excessive sympathetic activation in renovascular hypertensive rats.     

MAS受体与AT1受体的相互作用

RAS的2个重要成员Ang-(1-7)和血管紧张素II（Ang II）主要作用于MAS受体和AT1受体发挥作用。Ang-(1-7)和Ang II之间存在着复杂的相互作用，两者的受体在细胞和组织中相互作用，其可能的机制之一是受体的寡聚化     

Reactive oxygen species-mediated signaling pathways in angiotensin II-induced MCP-1 expression of proximal tubular cells

Angiotensin II (AngII) has pleiotropic effects, the most well known of which is the generation of reactive oxygen species (ROS) and chemokines in inflammatory lesions. Monocyte chemoattractant protein-1 (MCP-1) is considered a major chemokine in the pathogenesis of kidney diseases. We examined signaling pathways of AngII-induced MCP-1 expression and the role of ROS in the murine proximal tubular cells (mProx) using various inhibitors. Furthermore, we compared the signaling pathways between mProx and mesangial cells (MC). AngII-induced MCP-1 protein expression in mProx at 6 h was largely blocked by ROS (N-acetylcysteine; 82 +/- 14%), Ras (N-acetyl-S-trans,trans-farnesyl-L-cysteine; 82 +/- 13%), and nuclear factor-kappaB (NF-kappaB) (parthenolide; 89 +/- 7.9%) inhibitors. Both AT1 receptor (AT1R) (Olmesartan; 41 +/- 12%) and the AT2R (PD123319; 24 +/- 11%) antagonists partially blocked the MCP-1 expression. Furthermore, mitogen-activated protein kinase (MAPK) pathways were also implicated in this protein expression, but it is less dependent on ROS/Ras pathways. In MC, protein kinase (calphostin C; 84 +/- 2.8%) and NF-kappaB (89 +/- 1.4%) inhibitors attenuated acute AngII-induced MCP-1 expression stronger than ROS/Ras inhibitors (1.0 +/- 0.9/29 +/- 9.5%). MAPK pathways, especially p38 MAPK, were involved in MC more than in mProx. AT1R (69 +/- 8.6%) and AT2R (57 +/- 21%) antagonists also were blocked. We suggested that, although NF-kappaB activation has a critical role, signaling pathways are different between mProx and MC. ROS-mediated signaling in mProx may have more contribution to AngII-induced inflammatory responses than to those in MC.     

Renin angiotensin system modulates mTOR pathway through AT2R in HIVAN

Mammalian target of rapamycin (mTOR) has been reported to contribute to the development of HIV-associated nephropathy (HIVAN). We hypothesized that HIV may be activating renal tissue mTOR pathway through renin angiotensin system (RAS) via Angiotensin Receptor Type II receptor (AT2R). Renal tissues of Vpr transgenic and Tg26 (HIVAN) mice displayed enhanced phosphorylation of mTOR and p70S6K. Aliskiren, a renin inhibitor attenuated phosphorylation of both mTOR and p70S6K in renal tissues of HIVAN mice. Interestingly, Angiotensin Receptor Type I (AT1R) blockade did not modulate renal tissue phosphorylation of mTOR in HIVAN mice; on the other hand, AT2R blockade attenuated renal tissue phosphorylation of mTOR in HIVAN mice. In vitro studies, both renin and Ang II displayed enhanced mouse tubular cell (MTC) phosphorylation of p70S6K in a dose dependent manner. HIV/MTC also displayed enhanced phosphorylation of both mTOR and p70S6K; interestingly this effect of HIV was further enhanced by losartan (an AT1R blocker). On the other hand, AT2R blockade attenuated HIV-induced tubular cell phosphorylation of mTOR and p70S6K, whereas, AT2R agonist enhanced phosphorylation of mTOR and p70S6K. These findings indicate that HIV stimulates mTOR pathway in HIVAN through the activation of renin angiotensin system via AT2R.     

Activating auto-antibodies against the AT1 receptor in preeclampsia

Immune mechanisms have been shown to be important in the pathogenesis of preeclampsia. Here, we review our studies of agonistic antibodies against the AT1 receptor in the pathogenesis as well as a pathologic phenotype of this disorder, focusing on observations in our laboratory. We have demonstrated their specificity of the binding by Western blotting, co-localization, and co-immunoprecipitation experiments. AT1-AA induce signaling in vascular cells and trophoblasts including AP-1 and nuclear factor-kappaB (NF-kappaB) activation. The signaling results in tissue factor production and reactive oxygen species (ROS) generation, both of which have been implicated in preeclampsia. The role of AT1-AA in preeclampsia and other severe hypertensive conditions has not yet been proven with certainty. However, we believe the findings are compelling and warrant further study.