AT1 Receptor mRNA Research Articles

Elevated dietary sodium intake exacerbates myocardial hypertrophy associated with cardiac-specific overproduction of angiotensin II

Cardiac hypertrophy is an independent risk factor predictive of cardiovascular disease and is significantly associated with morbidity and mortality. The mechanism by which angiotensin II (Ang II) and dietary sodium exert additive effects on the development of cardiac hypertrophy is unclear. The goal of this study was to evaluate the hypothesis that, where there is a genetic predisposition to Ang II-dependent hypertrophy, there is also an increased susceptibility to sodium-induced hypertrophy mediated by AT1-receptor expression. Diets of low sodium (LS, 0.3% w:w) and high sodium (HS, 4.0% w:w) content were fed to adult (age 25 weeks) control wild-type mice (WT) and to transgenic mice exhibiting cardiac-specific overexpression of angiotensinogen (TG). At the conclusion of a 40-day treatment period, cardiac tissue weights were compared and the relative expression levels of Ang II receptor subtypes (AT(1A) and AT(2)) were evaluated using RT-PCR. WT and TG mice fed HS and LS diets maintained comparable weight gains during the treatment period. The normalised heart weights of TG mice were elevated compared to WT, and the extent of the increase was greater for mice maintained on the HS diet treatments (WT 12% vs TG 41% increase in cardiac weight index). While a similar pattern of growth was observed for ventricular tissues, the atrial weight parameters demonstrated an additional significant effect of dietary sodium on tissue weight, independent of animal generic type. No differences in the relative (GAPDH normalised) expression levels of AT(1A)- and AT(2)-receptor mRNA were observed between diet or animal generic groups. This study demonstrates that, where there is a pre-existing genetic condition of Ang II-dependent cardiac hypertrophy, the pro-growth effect of elevated dietary sodium is selectively augmented. In TG and WT mice, this effect was evident with a relatively short dietary treatment intervention (40 days). Evaluation of the levels of Ang II receptor mRNA further demonstrated that this differential growth response was not associated with an altered relative expression of either AT(1A)- or AT(2)-receptor subtypes. The cellular mechanistic bases for this specific ANG II-dietary sodium interaction remain to be elucidated.

Angiotensin II-dependent induction of AT(2) receptor expression after renal ablation.

Angiotensin (ANG) II can be associated with gene expression regulation. Thus we studied the possible role of ANG II in the regulation of AT(2) mRNA and protein expression. We utilized sham-operated renal ablation rats as well as renal ablation rats pretreated during the first 7 days of the development of renal damage with either the angiotensin-converting inhibitor ramipril, the AT(1) receptor antagonist losartan, or the AT(2) receptor antagonist PD-123319. Renal tissue was analyzed for histological changes and expression of AT(2) receptor mRNA (by RT-PCR) and protein (by immunohistochemistry). To explore the physiological role of AT(2) receptor overexpression in the development of renal damage, blood pressure, urinary protein excretion, and renal damage were evaluated. A time-dependent increase in the expression of AT(2) receptor mRNA and protein was observed at 7, 15, and 30 days after renal ablation. Because these effects were already evident at day 7, the effects of ramipril, losartan, or PD-123319 were tested at this time. The ramipril group and the PD-123319-pretreated group showed inhibition of AT(2) receptor expression, whereas the losartan-pretreated group showed a further increase in AT(2) receptor expression. Inhibition of the AT(2) receptor during renal ablation was associated with increased renal damage and a further increase in the blood pressure. This suggests that overexpression of AT(2) receptors after renal ablation is modulated by ANG II through its own AT(2) receptor and that functional expression of this effect may represent a counterregulatory mechanism to modulate the renal damage induced by renal ablation.

Changes in the expression of angiotensin II type 1 receptor in the development of liver fibrosis

To detect the expression of angiotensin II type 1 receptor (AT1R) in the different stages of human liver fibrosis. The morphology and ultrastructure of hepatic stellate cells (HSC) in hepatic sinusoids were studied by transmission electron microscopy, collagen I (col I) was tested by immunohistochemical method, an indirect immunofluorescence labeling method was used to detect AT1R, and semiquantitative RT-PCR was used to detect AT1R mRNA. Positive expression of AT1R was mainly distributed in the periphery of hepatic lobules and in the cytoplasm of HSC in the sinusoids. In normal liver tissue from 12 cases, positive expression of AT1R was seen in 8, whereas in 18 fibrotic livers, all showed positive expression. Cells that positively expressed AT1R were significantly more abundant in the fibrotic liver group than in the normal liver group (P < 0.001), and were significantly increased with an increase in the collagenous surface area. The relative expression level of AT1R mRNA in the fibrotic liver group was also higher than in the normal liver group (P < 0.01). The expressions of both AT1R and AT1R mRNA increased significantly with the degree of liver fibrosis, so angiotensin II and its receptor are probably important in the development of liver fibrosis.

ACE2 gene expression is up-regulated in the human failing heart.

BackgroundACE2 is a novel homologue of angiotensin converting enzyme (ACE). ACE2 is highly expressed in human heart and animal data suggest that ACE2 is an essential regulator of cardiac function in vivo. Since overactivity of the renin-angiotensin system contributes to the progression of heart failure, this investigation assessed changes in gene expression of ACE2, ACE, AT1 receptor and renin in the human failing heart.MethodsThe sensitive technique of quantitative reverse transcriptase polymerase chain reaction was used to determine the level of mRNA expression of ACE and ACE2 in human ventricular myocardium from donors with non-diseased hearts (n = 9), idiopathic dilated cardiomyopathy (IDC, n = 11) and ischemic cardiomyopathy (ICM, n = 12). Following logarithmic transformation of the data, a one-way analysis of variance was performed for each target gene followed by a Dunnett's test to compare the two disease groups IDC and ICM versus control.ResultsAs anticipated, ACE mRNA was found to be significantly increased in the failing heart with a 3.1 and 2.4-fold up-regulation found in IDC and ICM relative to non-diseased myocardium. Expression of ACE2 mRNA was also significantly up-regulated in IDC (2.4-fold increase) and ICM (1.8-fold increase) versus non-diseased myocardium. No change in angiotensin AT1 receptor mRNA expression was found in failing myocardium and renin mRNA was not detected.ConclusionsThese data suggest that ACE2 is up-regulated in human IDC and ICM and are consistent with the hypothesis that differential regulation of this enzyme may have important functional consequences in heart failure. This strengthens the hypothesis that ACE2 may be a relevant target for the treatment of heart failure and will hopefully spur further studies to clarify the functional effects in human myocardium of ACE2 derived peptides.

Stimulation of cyclic GMP production via AT2 and B2 receptors in the pressure-overloaded aorta after banding.

Abdominal aortic banding induces upregulation of the angiotensin II (Ang II) type-2 (AT2) receptor, thereby decreasing the contractile response to Ang II in the thoracic aorta of the rat. The aim of this study was to use a mouse model to clarify the mechanisms by which the banding elicits upregulation of the aortic AT2 receptor and the subsequent attenuation of Ang II responsiveness. Concomitantly with the elevation in blood pressure and plasma renin concentration after banding, AT2-receptor mRNA levels in the thoracic aorta rapidly increased in mice within 4 days. Upregulation of the AT2 receptor, as well as blood pressure elevation after banding, was abolished by losartan administration. The contractile response to Ang II was depressed in aortic rings of banding mice but not of sham mice, and was restored by either the AT2-receptor antagonist PD123319 or the bradykinin B2-receptor antagonist icatibant. cGMP content in the thoracic aorta of banding mice was 9-fold greater than that of sham mice, and the elevation was reduced to sham levels 1 hour after intravenous injection of PD123319 or icatibant. When aortic rings were incubated with Ang II, cGMP content increased in banding rings but not in sham rings; the pretreatment with PD123319 or icatibant inhibited Ang II-induced cGMP production. These results suggest that aortic banding induces upregulation of the AT2 receptor through increased circulating Ang II via the AT1 receptor, thereby activating a vasodilatory pathway in vessels through the AT2 receptor via the kinin/cGMP system.

1000. Introns of the Angiotensin II Type 2 Receptor (AT2) Enhance Its Adenoviral-Mediated Over-Expression In Vitro

Top of pageAbstract The angiotensin II type 2-receptor (AT2) has been implicated to play a role in cardiovascular, brain and renal function as well as in the modulation of various biological processes involved in development, cell differentiation, tissue repair and apoptosis. However, it's specific roles in these processes are far from established. In this study we have constructed two first generation recombinant adenoviral vectors that may be used to investigate the biological functions of the AT2: Ad5-G-AT2-GFP containing genomic AT2 (G-AT2) DNA with two introns and Ad5-C-AT2-GFP with AT2 cDNA (c-AT2). The foreign genes G-AT2, C-AT2 and GFP were driven separately by a CMV promoter. Primary cortical neuronal cultures, which contain low or undetectable levels of endogenous AT2, were grown for 7-10 days in 12-well plates. At this time, viral particles were added directly to the growth media (1×107 infectious units per well), and 3 days later the expression of AT2 or GFP were analyzed by immunocytochemistry, 125SI Angiotensin II binding, real-time PCR or Western blot. Transduction of neuronal cultures with Ad5-G-AT2-GFP led to a 41.6 fold higher AT2 expression than with Ad5-C-AT2-GFP, as determined by 125SI Angiotensin II binding. Real-time RT-PCR analysis showed AT2 mRNA level in neuronal culture transduced with Ad5-G-AT2-GFP was 40.8 fold higher than with Ad-C-AT2-GFP. In addition, a greater number of neurons in the Ad5-G-AT2-GFP transduced neuronal culture showed green fluorescence compared with Ad5-C-AT2-GFP transduced neuronal culture. By contrast, the expression of total GFP in Ad5-G-AT2-GFP transduced neuronal cultures was significantly lower than Ad5-C-AT2-GFP, according to Western blot data. Moreover, the green fluorescence of non-neuronal cells in the Ad5-C-AT2-GFP transduced neuronal cultures was much stronger than obtained with Ad5-G-AT2-GFP. In conclusion, the presence of the introns of the AT2 increased it's expression, at the transcriptional level, in primary neuronal cultures transduced with an AT2 recombinant Ad5 and inhibited the expression of GFP which was driven by another CMV promoter in the same recombinant Ad5 vector. Thus the increased over-expression obtained with the presence of AT2 introns may allow the use of lower numbers of viral particles, circumventing the problems of immunological toxicity obtained with Ad vectors.

Cerebral ischemia enhances vascular angiotensin AT1 receptor-mediated contraction in rats.

The aim of the study was to examine how focal cerebral ischemia affects the expression and function of vascular angiotensin II receptors. We used an intraluminal filament occlusion technique to occlude the right middle cerebral artery (MCA) of the rat. Myographs were used for functional studies of the MCA and real-time polymerase chain reaction, for determination of relative mRNA levels. The contractile responses to angiotensin II were stronger in the right occluded MCA compared with the left MCA and the MCA from sham-operated rats 48 hours after MCA occlusion (P<0.05). The angiotensin II type 1 (AT1) receptor antagonists candesartan and losartan abolished the enhanced responses to angiotensin II (P<0.05), whereas the AT2 receptor antagonist PD123319 had no effect. The amount of AT1 receptor mRNA was lower in the occluded MCAs compared with nonoccluded MCAs 48 hours after occlusion (P<0.05), whereas the mRNA levels of angiotensin converting enzyme (ACE) were higher in the occluded arteries. The mRNA levels of the AT2 receptor and nuclear factor-kappaB were unchanged. Focal cerebral ischemia in the rat upregulated the contractile responses to angiotensin II in the ipsilateral MCA, and this contraction was mediated by AT1 receptors. Real-time polymerase chain reaction revealed decreased AT1 receptor mRNA levels in the occluded MCA, whereas the amount of ACE mRNA was increased, suggesting locally enhanced angiotensin II production. These results support a role for AT1 receptors in cerebral ischemia, and we think that AT1 receptors might be a future therapeutic target in ischemic stroke.

Effect of Sodium on Vasoconstriction and Angiotensin II Type 1 Receptor mRNA Expression in Cold‐induced Hypertensive Rats

Angiotensin II and sodium play an important pathogenetic role in several models of hypertension. Now, we investigated the effects of sodium on vasoconstriction and angiotensin II type 1 (AT1) and type 2 (AT2) receptor mRNA expression in aortic vessels from cold‐induced hypertensive rats. Wistar rats on low sodium and high sodium diet were exposed to cold‐stress for 8 weeks. The effects of angiotensin II infusion on mean arterial blood pressure were investigated in these rats. In addition, angiotensin II induced contraction was measured using aortic rings. Expression of AT1 receptor mRNA and AT2 receptor mRNA was assessed in aortic vessels by reverse transcription polymerase chain reaction. After infusion of angiotensin II mean arterial blood pressure in cold‐induced hypertensive rats on high sodium diet was significantly higher compared to cold‐induced hypertensive rats on low sodium diet (p < 0.05). Angiotensin II‐induced contraction of aortic rings was significantly higher in cold‐induced hypertensive rats on high sodium diet compared to cold‐induced hypertensive rats on low sodium diet (2.39 ± 0.03 g vs. 2.21 ± 0.04 g, n = 12, p < 0.01). Angiotensin AT1 receptor mRNA was significantly higher in cold‐induced hypertensive rats on high sodium diet compared to cold‐induced hypertensive rats on low sodium diet (p < 0.05). It is concluded that in this nongenetic, nonsurgical animal model of cold‐induced hypertension increased vasoconstriction and increased AT1 receptor mRNA expression in aortic vessels are dependent on sodium intake.

Interleukin-6 induces oxidative stress and endothelial dysfunction by overexpression of the angiotensin II type 1 receptor.

Angiotensin II type 1 (AT1) receptor activation as well as proinflammatory cytokines such as interleukin-6 (IL-6) are involved in the development and progression of atherosclerosis. The detailed underlying mechanisms including interactions between inflammatory agonists and the renin-angiotensin system are poorly understood. Stimulation of cultured rat aortic vascular smooth muscle cells (VSMCs) with IL-6 led to upregulation of AT1 receptor mRNA and protein expression, as assessed by Northern and Western blot experiments. Nuclear run-on and transcription blockade experiments showed that IL-6 increases AT1 receptor mRNA de novo synthesis but not mRNA stability. Preincubation of VSMCs with IL-6 resulted in an enhanced angiotensin II-induced production of reactive oxygen species, as assessed by DCF fluorescence laser microscopy. Treatment of C57BL/6J mice with IL-6 for 18 days increased vascular AT1 receptor expression (real-time RT-PCR) and angiotensin II-induced vasoconstriction, enhanced vascular superoxide production (L-012 chemiluminescence, DHE fluorescence), and impaired endothelium-dependent vasodilatation. These effects were completely omitted in AT1 receptor knockout mice (AT1A-/- mice). Upregulation of vascular AT1 receptor expression in vitro and in vivo is decisively involved in IL-6-induced propagation of oxidative stress and endothelial dysfunction. This interaction of the proinflammatory cytokine IL-6 with the renin-angiotensin system may represent an important pathogenetic mechanism in the atherosclerotic process.

Effect of sodium on blood pressure, cardiac hypertrophy, and angiotensin receptor expression in rats

Background Sodium-induced hypertensive cardiac hypertrophy is related to pressure and volume overload. Methods Wistar rats were exposed to low and high sodium diet for 8 weeks. Angiotensin II receptor mRNA, abundance of p38 mitogen-activated protein kinase (p38MAPK), vasoconstriction of aortic rings, and angiotensin II-induced calcium increase were investigated. Results Rats on high sodium diet showed significantly elevated blood pressure. Heart weight, AT1 receptor mRNA in cardiac and aortic tissues, and abundance of p38MAPK were significantly increased in rats on high sodium diet. Conclusions Increased AT1 receptor expression and angiotensin II-induced calcium increase are compensatory effects in sodium-induced hypertension.

MRNA-binding proteins involved in the nucleo-cytosolic transfer of the human AT1 receptor mRNA in vascular smooth muscle cells

mRNA-binding proteins involved in the nucleo-cytosolic transfer of the human AT1 receptor mRNA in vascular smooth muscle cells

Angiotensin II regulates 11β-hydroxysteroid dehydrogenase type 2 via AT2 receptors

In preeclampsia, cortisol degradation by the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is compromised, which enhances intracellular cortisol availability. This leads to vasoconstriction and renal sodium retention with volume expansion, thus increasing blood pressure. An augmented availability of angiotensin II (Ang II) predisposes to preeclampsia. Some effects of Ang II are mediated by the mitogen-activated protein kinase (MAPK) cascade, which also regulates 11beta-HSD2 activity. Therefore, we hypothesized that Ang II regulates 11beta-HSD2. The human choriocarcinoma cell line JEG-3, which expresses the 11beta-HSD2 isoenzyme, was used. 3H-cortisol/cortisone conversion assays and mRNA analyses by reverse transcription-polymerase chain reaction (RT-PCR) were performed. Cells were stimulated with Ang II and the effect was modulated by Ang II type 1 (AT1) and AT2 receptor blockers DUP753 or L-158809 and PD-123319, respectively. In order to elucidate the signaling cascade, the MAPK kinase inhibitors PD-098059 and U-0126 were probed. The impact of a modulated 11beta-HSD2 activity was assessed by determining the effect of cortisol on AT1 receptor mRNA. Ang II reduced mRNA and activity of 11beta-HSD2 mainly by a post-transcriptional mechanism. This Ang II effect was abrogated by AT2, but not by AT1 receptor blockade. Mitogen-activated protein (MAP) kinase kinase (MAPKK) inhibitors reversed the Ang II effect. Dexamethasone augmented the mRNA expression of AT1 receptors. Cortisol enhanced AT1 receptor mRNA expression when the 11beta-HSD2 activity was reduced either by Ang II or by glycyrrhetinic acid, an 11beta-HSD2 inhibitor. Ang II decreases the activity of 11beta-HSD2 by an AT2 receptor- and MAPK-dependent mechanism. The decreased activity of 11beta-HSD2 increases the intracellular availability of cortisol, which might be relevant for the pathogenesis of hypertension and preeclampsia.

Angiotensin II mediates LDL-induced superoxide generation in mesangial cells.

Lipid abnormalities and activation of the local renin-angiotensin system (RAS) may be involved in the pathogenesis of chronic glomerular disease. This study investigated whether low-density lipoprotein (LDL) activates local RAS in cultured human mesangial cells (HMC) and, at the same time, whether ANG II mediates LDL-induced mesangial cell proliferation, hypertrophy, and superoxide (O2-) generation. Quiescent HMC were exposed to 50 to 200 microg/ml of LDL or 10-7 to 10-10 M ANG II for 0.5 to 24 h in the presence or absence of 10-6 M losartan, an ANG II type I (AT1) receptor antagonist, or 10-5 M diphehylendieodonium (DPI) or 10-4 M apocynin, inhibitors of nicotinamide adenine dinucleotide phosphate oxidase. LDL induced an up to threefold increase in the ANG II levels in the culture medium of HMC. LDL upregulated AT1 receptor and angiotensinogen mRNA expression in HMC. LDL incubated with HMC increased O2- production by up to 3.3 times compared with the level of control cells. The LDL-induced, increased O2- generation was suppressed by losartan, DPI, or apocynin. LDL significantly increased mesangial [3H]thymidine or [3H]leucine incorporation, whereas these processes were abrogated by losartan. In conclusion, LDL increases ANG II production by mesangial cells, which in turn results in increased O2- production, and cell proliferation and hypertrophy, these effects of ANG II being mediated by the AT1 receptor.

L-arginine reverses renin, hypertension, and electrolyte excretion changes in pregnancy induced hypertensive rats

Pregnancy-induced hypertensive (PIH) disorders are a leading cause of maternal mortality and fetal morbidity. Spontaneous sustained hypertension and intrauterine growth restriction often characterize these disorders. The SHHF/Mcc-facp (SHHF) has been used as an animal model of the human disorders because it has the above features plus abnormal gene expression in the placental and kidney. Although the mechanisms responsible for PIH are unknown, altered function of the renin-angiotensin system (RAS) has been implicated. To gain a molecular understanding of the role of the RAS in spontaneous PIH in SHHF rats, kidney mRNA expression profile of RAS components at gestation day 20 were compared with normotensive pregnant WKY controls. By gestation day 10, systolic blood pressure was 40 ± 5 mm Hg higher than pre-pregnant values compared to hypotension in pregnant WKY and remained elevated for the duration of pregnancy. Na and Ca excretion were lower than WKY controls despite higher blood pressure. PRA was significantly lower in SHHF rats (2.1 ± 0.1 ng/ml.hr vs 9.7 ± 1.3 ng/ml.hr, p<0.05) and renin mRNA was downregulated 66± 7% compared to pregnant WKY. Although AGT mRNA was undetectable in SHHF rat kidney, ACE RnBP, and AT1 receptor were all highly expressed. L-arginine administration beginning on day 1 reversed changes in blood pressure, Na and Ca excretion, as well as renin mRNA expression, suggesting that there may be an association between Na, Ca, and renin in PIH. L-arginine did not alter abundance of AGT, RnBP, ACE, or AT1receptor mRNA, suggesting that regulation of renin expression and Na and Ca expression in PIH rats may be mediated by the NO pathway. We conclude that the impaired RAS in PIH rats is at the level of renin gene expression and that this impairment may be reversed by L-arginine.

A role for angiotensin II AT1 receptors in ureteric bud cell branching.

Gene-targeting studies in mice demonstrate that the renin-angiotensin system is required for the proper development of the renal medulla. In the absence of angiotensin II (ANG II) or the ANG II type 1 (AT1) receptor, mice exhibit poor papillary development and a severe urinary-concentrating defect. These findings imply that the ureteric bud (UB) and its branches are targets for ANG II actions during renal development. However, direct evidence linking ANG II with UB-branching morphogenesis does not exist. Using immunohistochemistry, we demonstrated that UB-derived epithelia express angiotensinogen (Ao) and the AT1 receptor during murine metanephrogenesis. Ao and AT1 receptors are expressed in the UB branches and to a lesser extent in the stromal mesenchyme. AT1 receptor expression in UB-derived epithelia increased from embryo day 12 to day 16 and was observed on both luminal and basolateral membranes. In accord with these findings, cultured murine UB cells express AT1 receptor protein and mRNA. Treatment of UB cells cultured in three-dimensional type I collagen gels with ANG II (10-7 to 10-5 M) elicits a dose-related increase in the number of cells that have primary and secondary branches. These effects of ANG II on UB branching are abrogated by pretreatment with the AT1 receptor antagonist candesartan. These data demonstrate a direct and independent role for ANG II acting via AT1 receptors on UB cell branching in vitro. The presence of Ao in the stroma and AT1 on UB cells supports the notion that cross talk between stroma and epithelial cells is crucial to epithelial branching morphogenesis in the developing kidney.

Effects of mitogen-activated protein kinase pathway and co-activator CREP-binding protein on peroxisome proliferator-activated receptor-gamma-mediated transcription suppression of angiotensin II type 1 receptor gene.

Peroxisome proliferator-activated receptor (PPAR)-gamma and its ligands suppress several genes related to atherogenesis. We previously reported that ligand-activated PPAR-gamma suppressed angiotensin II type 1 receptor (AT1R) gene transcription in vascular smooth muscle cells (VSMCs) by the inhibition of Sp1 binding to the --58/--34 GC-box related element in the AT1R gene promoter region via a protein-protein interaction. It has been reported that the mitogen-activated protein (MAP) kinase pathway inhibits PPAR-gamma function through its phosphorylation, and co-activator CREB-binding protein (CBP)/p300 interacts with PPAR-gamma and modulates its activity. Since both the MAP kinase pathway and CBP have recently been reported to be atherogenic, we examined their effects on PPAR-gamma-mediated AT1R gene transcription suppression. We observed that 1) PPAR-gamma-mediated AT1R gene transcription suppression was augmented by treatment with the MAP kinase kinase inhibitor PD98059, while treatment with the p38 kinase inhibitor SB203580 showed no effect; 2) the PPAR-gamma-mediated AT1R mRNA decrease was also augmented by PD98059 treatment; 3) CBP overexpression partially, but significantly, abrogated PPAR-gamma-mediated AT1R gene transcription suppression; and 4) the CBP effect was eliminated when the --58/--34 GC-box related element was disrupted. It is therefore speculated that: 1) PPAR-gamma phosphorylation by the MAP kinase pathway may attenuate PPAR-gamma-mediated AT1R gene transcription suppression through the inhibition of PPAR-gamma activity; and 2) CBP may enhance the activity of the remaining Sp1 on the --58/--34 GC-box related element, resulting in a reduction in PPAR-gamma-mediated AT1R gene transcription suppression. The MAP kinase pathway and CBP may thus antagonize against PPAR-gamma in AT1R gene transcription, probably leading to the progression of atherosclerosis.

The Role of the Renin-Angiotensin System in Cholesterol and Puromycin Mediated Renal Injury

Puromycin aminonucleoside (PAN) nephropathy is a widely studied model of glomerular sclerosis (GS) in the rat, and cholesterol feeding exacerbates the injury induced by PAN. The importance of the interaction of angiotensin II (Ang II) with the AT2 receptor is unclear. We investigated the role of the renin-angiotensin system, particularly with regard to AT1 and AT2 receptor dynamics, in PAN and cholesterol-mediated GS. Sprague-Dawley rats were given a 4% cholesterol diet (group II), subcutaneous PAN (group III), or a 4% cholesterol diet and PAN (group IV) and compared with a control group given PAN vehicle (group I). After 16 weeks, kidneys were harvested and tissue Ang II concentration, angiotensin-converting enzyme (ACE) activity, and ACE, AT1, and AT2 mRNA levels were determined. Compared with control rats, proteinuria was significantly higher in groups II to IV. Kidney ACE activity and ACE mRNA levels in groups III and IV were 2- and 3-fold higher than in groups I and II, respectively. Kidney Ang II concentration also was increased in the experimental groups. Whereas kidney AT1 mRNA was significantly lower in groups III and IV, kidney AT2 mRNA was significantly increased in groups II to IV. In these experimental models of GS, there is significant activation of the tissue-based renin-angiotensin system. Puromycin with and without cholesterol decreased the AT1 receptor mRNA and increased the AT2 receptor mRNA. Up-regulation of AT2 receptors may be important in ameliorating the proliferative effects of Ang II, which presumably occur through the AT1 receptor.

Evidence of Apoptosis: Bax and Fas Expression in Acute Phase of Myocardial Infarction in Rats

Myocardial infarction (MI) remains the leading cause of death from cardiovascular diseases over the past decades. Apoptosis is ‘programmed’ cell death which leads to clearance of ‘unwanted’ cells without disruption of tissue structure or function. Recently, we reported that angiotensin receptor subtype AT1 and AT2 mRNA levels were time dependently regulated after MI. AT1 and AT2 receptor mRNA levels markedly increased at 30 min and peaked 24 h post-MI. The time-dependent increase of AT1 and AT2 receptor mRNA is associated with the early remodeling process of the non-infarcted myocardium post MI. There is a further question raised whether the up-regulation of AT2 receptor is linked to apoptosis. Therefore, we investigated the apoptotic development and apoptotic related gene expression after MI at different time points in the current study. MI was induced in Wistar rats by ligation of the left anterior descending coronary artery. Bax gene expression was found to be increased at 12 h after MI and peaked at 24 h (4.3-fold). It declined at 72 h after MI. Fas gene started to over-express at 12 h after MI as well but it reached maximum at 72 h (4.7-fold). Protein levels of Bax and Fas expression were detected in the necrotic area and area at risk (surrounding area) at different time points after MI. Apoptosis was detected in the infarcted areas. No apoptosis was detected in the sham operated rats. In the MI groups, strongest staining of apoptosis was detected in rats 3 days post operation. Weak staining was found 1 day, 7 day post MI. Very fewer apoptotic cells were detected in the rats 2 weeks after MI. Our results demonstrate that apoptotic development after MI is time dependent in the ischemic area and there could be some linkage with the over expression of AT2 receptor post MI.

Angiotensin-(1-7) inhibits angiotensin II-induced signal transduction.

The inhibitory effects of angiotensin-(1-7) on angiotensin II-induced vasoconstriction, growth of vascular smooth muscle cells, stimulation of protein kinase C, extracellular signal-regulated kinases (ERK), and angiotensin subtype 1 receptor (AT1) and subtype 2 receptor (AT2) mRNA expression were investigated. The hemodynamic effects of angiotensin-(1-7) were measured in Wistar rats. Vasoconstriction was measured using aortic rings. DNA synthesis or protein synthesis was measured in cultured vascular smooth muscle cells using [3H] thymidine or [3H] leucine incorporation, respectively. Angiotensin II stimulated protein kinase C and ERK1/2 were measured by Western blot analysis using phosphospecific protein kinase C and ERK1/2 antibodies. AT1 and AT2 receptor mRNA expression was measured using reverse-transcription polymerase chain reaction. Infusion of angiotensin II significantly increased whereas infusion of angiotensin-(1-7) had no effects on mean arterial blood pressure in Wistar rats. Angiotensin-(1-7) dose-dependently showed partial antagonism on angiotensin II-induced contraction of aortic rings. Angiotensin-(1-7) showed partial antagonism on angiotensin II-induced DNA synthesis and protein synthesis. Angiotensin-(1-7) showed partial antagonism on angiotensin II-induced activation of protein kinase C and ERK1/2. The administration of angiotensin-(1-7) showed partial antagonism on angiotensin II-induced downregulation of AT1 receptor mRNA expression, whereas AT2 receptor mRNA expression was unchanged. Angiotensin-(1-7) showed partial antagonism on angiotensin II-induced intracellular signal transduction and may play a crucial role in the adaptation process of AT1 receptors to sustained stimulation of angiotensin II.

Estrogen upregulates renal angiotensin II AT(2) receptors.

AT(2) receptors may act in opposition to and in balance with AT(1) receptors, their stimulation having beneficial effects. We found renal AT(2) receptor expression in female mice higher than in male mice. We asked the question of whether such expression might be estrogen dependent. In male, female, ovariectomized, and estrogen-treated ovariectomized mice, we studied renal AT(1) and AT(2) receptors by immunocytochemistry and autoradiography, AT(2) receptor mRNA by RT-PCR, and cAMP, cGMP, and PGE(2) by RIA. AT(1) receptors predominated. AT(2) receptors were present in glomeruli, medullary rays, and inner medulla, and in female kidney capsule. AT(1) and AT(2) receptors colocalized in glomeruli. Female mice expressed fewer glomerular AT(1) receptors. Ovariectomy decreased AT(1) receptors in medullary rays and capsular AT(2) receptors. Estrogen administration normalized AT(1) receptors in medullary rays and increased AT(2) receptors predominantly in capsule and inner medulla, and also in glomeruli, medullary rays, and inner stripe of outer medulla. In medullas of estrogen-treated ovariectomized mice there was higher AT(2) receptor mRNA, decreased cGMP, and increased PGE(2) content. We propose that the protective effects of estrogen may be partially mediated through enhancement of AT(2) receptor stimulation.