Angiotensin-converting Enzyme Protein Expression Research Articles

Sex-Specific Changes in Renal Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 Gene Expression and Enzyme Activity at Birth and Over the First Year of Life.

Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) are key enzymes of the renin-angiotensin system. We investigated developmental changes in renal ACE and ACE2 gene expression and activity in both male and female sheep. Three groups of sheep (fetus, newborn, and adult) were used. Renal ACE and ACE2 activities, messenger RNA (mRNA), and protein expression were studied. Renal ACE and ACE2 activities increased at 1 year in males, while there were no changes throughout development in females. Renal ACE and ACE2 mRNA and protein showed no sex differences but increased by 1 year of age. There are sex-related differences in the development of renal-converting enzyme activities that may have functional implications in terms of the regulation of blood pressure and renal function in men and women. The difference in the patterns of gene expression and enzyme activity indicates that changes in gene expression may not accurately reflect changes in activity.

Mesenchymal Stem Cells Overexpressing Angiotensin-Converting Enzyme 2 Rescue Lipopolysaccharide-Induced Lung Injury

Bone marrow-derived mesenchymal stem cells (MSCs), which have beneficial effects in acute lung injury (ALI), can serve as a vehicle for gene therapy. Angiotensin-converting enzyme 2 (ACE2), a counterregulatory enzyme of ACE that degrades angiotensin (Ang) II into Ang 1-7, has a protective role against ALI. Because ACE2 expression is severely reduced in the injured lung, a therapy targeted to improve ACE2 expression in lung might attenuate ALI. We hypothesized that MSCs overexpressing ACE2 would have further benefits in lipopolysaccharide (LPS)-induced ALI mice, when compared with MSCs alone. MSCs were transduced with ACE2 gene (MSC-ACE2) by a lentiviral vector and then infused into wild-type (WT) and ACE2 knockout (ACE2(-/y)) mice following an LPS-induced intratracheal lung injury. The results demonstrated that the lung injury of ALI mice was alleviated at 24 and 72 h after MSC-ACE2 transplantation. MSC-ACE2 improved the lung histopathology and had additional anti-inflammatory effects when compared with MSCs alone in both WT and ACE2(-/y) ALI mice. MSC-ACE2 administration also reduced pulmonary vascular permeability, improved endothelial barrier integrity, and normalized lung eNOS expression relative to the MSC group. The beneficial effects of MSC-ACE2 could be attributed to its recruitment into the injured lung and enhanced local expression of ACE2 protein without changing the serum ACE2 levels after MSC-ACE2 transplantation. The biological activity of the increased ACE2 protein decreased the Ang II amount and increased the Ang 1-7 level in the lung when compared with the ALI and MSC-only groups, thereby inhibiting the detrimental effects of accumulating Ang II. Therefore, compared to MSCs alone, the administration of MSCs overexpressing ACE2 resulted in a further improvement in the inflammatory response and pulmonary endothelial function of LPS-induced ALI mice. These additional benefits could be due to the degradation of Ang II that accompanies the targeted overexpression of ACE2 in the lung.

Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress

ObjectivesWe hypothesized that chronic ethanol intake enhances vascular oxidative stress and induces hypertension through renin–angiotensin system (RAS) activation. Methods and resultsMale Wistar rats were treated with ethanol (20% v/v). The increase in blood pressure induced by ethanol was prevented by losartan (10mg/kg/day; p.o. gavage), a selective AT1 receptor antagonist. Chronic ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels and serum aldosterone levels. No differences on plasma osmolality and sodium or potassium levels were detected after treatment with ethanol. Ethanol consumption did not alter ACE activity, as well as the levels of ANG I and ANG II in the rat aorta or mesenteric arterial bed (MAB). Ethanol induced systemic and vascular oxidative stress (aorta and MAB) and these effects were prevented by losartan. The decrease on plasma and vascular nitrate/nitrite (NOx) levels induced by ethanol was prevented by losartan. Ethanol intake did not alter protein expression of ACE, AT1 or AT2 receptors in both aorta and MAB. Aortas from ethanol-treated rats displayed decreased ERK1/2 phosphorylation and increased protein expression of SAPK/JNK. These responses were prevented by losartan. MAB from ethanol-treated rats displayed reduced phosphorylation of p38MAPK and ERK1/2 and losartan did not prevent these responses. ConclusionsOur study provides novel evidence that chronic ethanol intake increases blood pressure, induces vascular oxidative stress and decreases nitric oxide (NO) bioavailability through AT1-dependent mechanisms.

Activation of renin-angiotensin-aldosterone system (RAAS) in the lung of smoking-induced pulmonary arterial hypertension (PAH) rats.

Objectives:To explore the role of the renin–angiotensin–aldosterone system (RAAS) in the pathogenesis of pulmonary arterial hypertension (PAH) induced by chronic exposure to cigarette smoke.Methods:48 healthy male SD rats were randomly divided into four groups (12/group): control group (group A); inhibitor alone group (group B); cigarette induction group (group C); cigarette induction + inhibitor group (group D). After the establishment of smoking-induced PAH rat model, the right ventricular systolic pressure (RVSP) was detected using an inserted catheter; western blotting was used to detect the protein expression of angiotensin-converting enzyme-2 (ACE2) and angiotensin-converting enzyme (ACE); expression levels of angiotensin II (AngII) in lung tissue were measured by radioimmunoassay.Results:After six months of cigarette exposure, the RVSP of chronic cigarette induction group was significantly higher than that of the control group; expression levels of AngII and ACE increased in lung tissues, but ACE2 expression levels reduced. Compared with cigarette exposure group, after losartan treatment, RVSP, ACE and AngII obviously decreased (P<0.05), and ACE2 expression levels significantly increased.Conclusion:Chronic cigarette exposure may result in PAH and affect the protein expression of ACE2 and ACE in lung tissue, suggesting that ACE2 and ACE play an important role in the pathogenesis of smoking-induced PAH.

MSCs Modified With ACE2 Restore Endothelial Function Following LPS Challenge by Inhibiting the Activation of RAS

Angiotensin (Ang) II plays an important role in the process of endothelial dysfunction in acute lung injury (ALI) and is degraded by angiotensin-converting enzyme2 (ACE2). However, treatments that target ACE2 to injured endothelium and promote endothelial repair of ALI are lacking. Mesenchymal stem cells (MSCs) are capable of homing to the injured site and delivering a protective gene. Our study aimed to evaluate the effects of genetically modified MSCs, which overexpress the ACE2 protein in a sustained manner via a lentiviral vector, on Ang II production in endothelium and in vitro repair of lipopolysaccharide (LPS)-induced endothelial injury. We found that the efficiency of lentiviral vector transduction of MSCs was as high as 97.8% and was well maintained over 30 passages. MSCs modified with ACE2 showed a sustained high expression of ACE2 mRNA and protein. The modified MSCs secreted soluble ACE2 protein into the culture medium, which reduced the concentration of Ang II and increased the production of Ang 1-7. MSCs modified with ACE2 were more effective at restoring endothelial function than were unmodified MSCs, as shown by the enhanced survival of endothelial cells; the downregulated production of inflammatory mediators, including ICAM-1, VCAM-1, TNF-α, and IL-6; reduced paracellular permeability; and increased expression of VE-cadherin. These data demonstrate that MSCs modified to overexpress the ACE2 gene can produce biologically active ACE2 protein over a sustained period of time and have an enhanced ability to promote endothelial repair after LPS challenge. These results encourage further testing of the beneficial effects of ACE2-modified MSCs in an ALI animal model.

Angiotensin converting enzyme is involved in the cardiac hypertrophy induced by sinoaortic denervation in rats.

The present study was designed to test the hypothesis that local angiotensin converting enzyme (ACE) was involved in the cardiac hypertrophy induced by sinoaortic denervation (SAD) in rats. Experiment 1: Six weeks after SAD of rats, components of renin-angiotensin system (RAS) in left ventricles were assayed by quantitative real-time PCR and Western blotting analysis. Experiment 2: Rats were divided into five groups treated as follows: (1) sham-operated group; (2) SAD group; (3) SAD group treated with angiotensin II type 1 receptor (AT1R) antagonist losartan (10 mg·kg(-1)·day(-1), orally); (4) SAD group treated by ACE inhibitor ramipril (1 mg·kg(-1)·day(-1), orally); (5) SAD group treated by ramipril and the B2-kinin receptor selective antagonist HOE-140 (0.25 mg·kg(-1)·day(-1), subcutaneously). SAD led to augmentation of the mRNA levels and protein expression of left ventricular ACE and AT1R. Both losartan and ramipril ameliorated SAD-induced left ventricular hypertrophy. Both losartan and ramipril abated oxidative stress, suppressed inflammation, and reduced expression TGFβ-R in left ventricles. In addition, the protective effect of ramipril could be abolished by HOE-140. Local ACE is involved in the left ventricular hypertrophy induced by sinoaortic denervation in rats, via both angiotensin II/AT1R and bradykinin/B2R pathways.

Beneficial Effects of Renin-Angiotensin System Blockers on Testicular Steroidogenesis

We evaluated whether antihypertensive drugs that act through the renin-angiotensin system would affect testis function. Ten mice were fed standard chow and 40 received a high energy density diet. At 8 weeks the high energy density diet mice were divided into 4 groups of 10 each. The untreated group received the high energy density dietalone. The 3 treated groups received that diet plus aliskiren (50 mg/kg daily), enalapril (30 mg/kg daily) and losartan (10 mg/kg daily), respectively, for the next 6 weeks. Blood pressure was measured twice monthly. At the end of the treatment period all mice were sacrificed. One-way ANOVA and the Holm-Sidak post hoc test were used to analyze results. The high energy density diet led to a significant increase in blood pressure (p <0.05). All treatments resulted in normalized blood pressure. In regard to reproductive function, and serum testosterone and estradiol the gene and protein expression of StAR, aromatase and luteinizing hormone receptor, and the protein expression of angiotensin-converting enzyme, renin and angiotensin type 1 receptor blocker were significantly decreased by the high energy density diet. Of the treatments only enalapril reverted the changes. Also, angiotensin-converting enzyme, angiotensin type 1 receptor blocker and renin protein expression were lower in all high energy density diet groups except the group that received enalapril. Only angiotensin-converting enzyme inhibitor reverted the hormonal and testis alterations caused by the high energy density diet. This suggests that enalapril should be the drug of choice for a patient who presents with previous reproductive dysfunction.

Overexpression of angiotensin-converting enzyme 2 attenuates tonically active glutamatergic input to the rostral ventrolateral medulla in hypertensive rats.

The rostral ventrolateral medulla (RVLM) plays a key role in cardiovascular regulation. It has been reported that tonically active glutamatergic input to the RVLM is increased in hypertensive rats, whereas angiotensin-converting enzyme 2 (ACE2) in the brain has been suggested to be beneficial to hypertension. This study was designed to determine the effect of ACE2 gene transfer into the RVLM on tonically active glutamatergic input in spontaneously hypertensive rats (SHRs). Lentiviral particles containing enhanced green fluorescent protein (lenti-GFP) or ACE2 (lenti-ACE2) were injected bilaterally into the RVLM. Both protein expression and activity of ACE2 in the RVLM were increased in SHRs after overexpression of ACE2. A significant reduction in blood pressure and heart rate in SHRs was observed 6 wk after lenti-ACE2 injected into the RVLM. The concentration of glutamate in microdialysis fluid from the RVLM was significantly reduced by an average of 61% in SHRs with lenti-ACE2 compared with lenti-GFP. ACE2 overexpression significantly attenuated the decrease in blood pressure and renal sympathetic nerve activity evoked by bilateral injection of the glutamate receptor antagonist kynurenic acid (2.7 nmol in 100 nl) into the RVLM in SHRs. Therefore, we suggest that ACE2 overexpression in the RVLM attenuates the enhanced tonically active glutamatergic input in SHRs, which may be an important mechanism underlying the beneficial effect of central ACE2 to hypertension.

Urinary ACE2 in healthy adults and patients with uncomplicated type 1 diabetes.

Angiotensin-converting enzyme 2 (ACE2) is expressed in the kidney and may be renoprotective. We determined whether urinary ACE2 enzyme activity and protein levels (ELISA), as well as angiotensinogen and ACE, are elevated during clamped euglycemia (4-6 mmol·L(-1)) in patients with uncomplicated type 1 diabetes (T1D, n = 58) compared with normoglycemic controls (n = 21). We also measured the effect of clamped hyperglycemia (9-11 mmol·L(-1)) on each urinary factor in T1D patients. Urinary ACE2 activity and protein levels were higher during clamped euglycemia in T1D compared with the controls (p < 0.0001). In contrast, urinary angiotensinogen levels (p = 0.27) and ACE excretion (p = 0.68) did not differ. In response to clamped hyperglycemia in T1D, urinary ACE2 protein decreased (p < 0.0001), whereas urinary ACE2 activity as well as angiotensinogen and ACE levels remained unchanged. Urinary ACE2 activity and protein expression are increased in T1D patients prior to the onset of clinical complications. Further work is required to determine the functional role of urinary ACE2 in early T1D.

Expression of angiotensin-converting enzyme gene in whole blood in patients with essential hypertension

Objective: The present study aims to investigate the correlation of the angiotensin-converting enzyme (ACE) gene expression and protein expression in patients with essential hypertension in whole blood.Methods: ACE gene expression was analyzed by Real Time PCR and western blot in 52 patients with essential hypertension and 42 healthy controls.Results: We observed a significant increase in Delta threshold cycle (ΔCT) values in the circulating ACE gene and ACE protein expression in patients as compared to controls.Conclusions: The up-regulation in relative expression of circulating Angiotensin converting enzyme mRNA and protein in patients with respect to controls might be correlated with high blood pressure in patients with essential hypertension.

Albumin inhibits the insulin-mediated ACE2 increase in cultured podocytes

Podocytes are key cells in the glomerular filtration barrier with a major role in the development of diabetic nephropathy. Podocytes are insulin-sensitive cells and have a functionally active local renin-angiotensin system. The presence and activity of angiotensin-converting enzyme 2 (ACE2), the main role of which is cleaving profibrotic and proinflammatory angiotensin-II into angiotensin-(1-7), have been demonstrated in podocytes. Conditionally immortalized mouse podocytes were cultured with insulin in the presence and absence of albumin. We found that insulin increases ACE2 gene and protein expression, by real-time PCR and Western blotting, respectively, and enzymatic activity within the podocyte and these increases were maintained over time. Furthermore, insulin favored an "anti-angiotensin II" regarding ACE/ACE2 gene expression balance and decreased fibronectin gene expression as a marker of fibrosis in the podocytes, all studied by real-time PCR. Similarly, insulin incubation seemed to protect podocytes from cell death, studied by a terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. However, all these effects disappeared in the presence of albumin, which may mimic albuminuria, a main feature of DN pathophysiology. Our results suggest that modulation of renin-angiotensin system balance, fibrosis, and apoptosis by insulin in the podocyte may be an important factor in preventing the development and progression of diabetic kidney disease, but the presence of albuminuria seems to block these beneficial effects.

Abstract 159: Inhibition of Ace Does Not Prevent 20-hete-mediated Vascular Injury in Cyp4a12tg Mice

20-hydroxyeicosatetraenoic acid (20-HETE), a cytochrome P450-arachidonic acid metabolite in the microcirculation, increases smooth muscle contraction and proliferation, stimulates endothelial dysfunction and activation and promotes hypertension that is dependent, in part, on the renin angiotensin system (RAS). We showed that in cultured human microvascular endothelial cells, 20-HETE increases angiotensin converting enzyme (ACE) mRNA by 3.1-fold (± 0.16; p&lt;0.05), ACE protein by 4.2-fold (±0.99; p&lt;0.05) and cellular and extracellular ACE activity by 2.2-fold (±0.09; p&lt;0.05) and 1.96-fold (±0.16; p&lt;0.05), respectively. The contribution of ACE to vascular 20-HETE-mediated microvascular remodeling in hypertension was assessed using the Cyp4a12 transgenic mouse in which the expression of the Cyp4a12-20-HETE synthase is driven by a tetracyclin (doxycycline, DOX)-sensitive promoter. Treatment of Cyp4a12tg mice with DOX increased blood pressure (133±6 vs. 107±3 mmHg; p&lt;0.05) and remodeling of renal resistance arteries (M/L, 0.15±0.01 vs. 0.07±0.01; p&lt;0.05); these increases were abrogated by co-treatment with the 20-HETE antagonist, 20-HEDGE. Renal ACE protein expression was significantly higher in DOX-treated (1.6 ± 0.17 fold; p&lt;0.05) as compared to untreated Cyp4a12tg mice; co-treatment with 20-HEDGE prevented the increase in ACE expression. Lisinopril treatment of Cyp4a12tg mice concurrently receiving DOX prevented the blood pressure increase (133±6 vs. 104±2 mmHg; p&lt;0.05) but did not interfere with renal microvascular 20-HETE production (1.05±0.18 vs 1.09±0.30 ng/mg). Co-treatment with lisinopril and DOX attenuated but did not prevent remodeling of renal microvessels (M/L, 0.07±0.0 vs. 0.15±0.01 vs. 0.11±0.01; p&lt;0.05 in water, DOX and DOX+Lisinopril, respectively). In contrast, co-treatment with 20-HEDGE prevented DOX-induced remodeling of resistance arteries of Cyp4a12tg mice. This study demonstrates that 20-HETE is a key determinant of microvascular remodeling in DOX-induced Cyp4a12tg hypertensive mice. 20-HETE’s mechanism only partially relies on activation of the RAS. Thus, the 20-HETE-driven microvascular remodeling does not fully depend on the upregulation of ACE and elevation of blood pressure.

Angiotensin‐Converting Enzyme Inhibition and Angiotensin AT1 Receptor Blockade Downregulate Angiotensin‐Converting Enzyme Expression and Attenuate Renal Injury in Streptozotocin‐Induced Diabetic Rats

Angiotensin-converting enzyme (ACE) is upregulated in the diabetic kidney and contributes to renal injury. This study investigates the possible beneficial effects of the ACE inhibitor (ACEI), enalapril and the AT1 receptor blocker (ARB), valsartan, on renal ACE expression, renal structure, and function in streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were allocated into four groups: control, STZ-diabetic rats, and STZ-diabetic rats treated with either enalapril (10 mg/kg/day) or valsartan (50 mg/kg/day) for 8 weeks. Enalapril and valsartan reduced renal ACE mRNA and protein expression, Na(+) /K(+) -ATPase activity, oxidative stress, and serum transforming growth factor-β1 levels compared to the diabetic group. Both treatments normalized renal nitrate/nitrite levels and ameliorated the observed histopathological changes. In conclusion, ACE downregulation by ACEI and ARB indicates that angiotensin II upregulates ACE through AT1 receptor. Prevention of diabetes-induced changes in ACE expression and Na(+) /K(+) -ATPase activity could be a new explanation of the renoprotective effects of ACEIs and ARBs.

Effects of rosuvastatin on expression of angiotensin-converting enzyme 2 after vascular balloon injury in rats.

ObjectiveTo investigate the effects and mechanisms of rosuvastatin on angiotensin -converting enzyme 2 (ACE2) in the process of neointimal formation after vascular balloon injury in rats, and to explore the effects of ACE2 and rosuvastatin in restenosis.MethodsThirty-six Wistar rats were randomly allocated into three groups: control group (n = 12), surgery group (n = 12), and statin group (n = 12). Aortic endothelial denudation of rats was performed using 2F balloon catheters. At days 14 and 28 after injury, aortic arteries were harvested to examine the following. Intimal thickening was examined by hematoxylin and eosin staining. We measured angiotensin II (Ang II) and angiotensin 1-7 (Ang-[1–7]) levels by a radioimmunological method or enzyme-linked immunosorbent assay. Protein and mRNA expression of ACE2 and Ang II type 1 receptor (AT1) were investigated by immunohistochemistry, Western blots, and Reverse transcriptase-polymerase chain reaction (RT-PCR). We measured changes in proliferating cell nuclear antigen (PCNA) by immunohistochemistry. The level of phosphorylated extracellular signal regulated kinase 1/2 (P-ERK1/2) was evaluated by Western blotting.ResultsProliferation of vascular smooth muscle cells (VSMC) and intimal thickening were higher at day 14 after vascular balloon injury in the surgery group compared with the control group. Proliferation of VSMC was decreased by day 28 after injury, while intimal thickening continued. With rosuvastatin treatment, the extent of VSMC proliferation and intimal thickening was reduced at day 14 and 28 after injury. Ang II and P-ERK levels were significantly increased, Ang-(1–7) levels were significantly decreased, mRNA and protein expressions of ACE2 were significantly decreased, and AT1 expression was significantly increased at days 14 and 28 after vascular balloon injury in the surgery group compared with the control group. PCNA expression was higher in the surgery group than in the control group, and it was significantly decreased after being given rosuvastatin. Expression of ACE2 mRNA and protein, and Ang-(1–7) levels were significantly increased, while AT1 expression and levels of Ang II and P-ERK were significantly decreased in the statin group compared with the surgery group.ConclusionsExpression of ACE2 mRNA and protein is decreased in the process of intimal thickening after balloon injury. The inhibitory effect of rosuvastatin on intimal thickening is related to upregulation of ACE2, an increase in Ang-(1–7), downregulation of AT1, and activation of the P-ERK pathway.

Rosiglitazone Treatment of Type 2 Diabetic db/db Mice Attenuates Urinary Albumin and Angiotensin Converting Enzyme 2 Excretion

Alterations within the renal renin angiotensin system play a pivotal role in the development and progression of cardiovascular and renal disease. Angiotensin converting enzyme 2 (ACE2) is highly expressed in renal tubules and has been shown to be renoprotective in diabetes. The protease, a disintegrin and metalloprotease (ADAM) 17, is involved in the ectodomain shedding of several transmembrane proteins including ACE2. Renal ACE2 and ADAM17 were significantly increased in db/db mice compared to controls. We investigated the effect of the insulin sensitizer, rosiglitazone, on albuminuria, renal ADAM17 protein expression and ACE2 shedding in db/db diabetic mice. Rosiglitazone treatment of db/db mice normalized hyperglycemia, attenuated renal injury and decreased urinary ACE2 and renal ADAM17 protein expression. Urinary excreted ACE2 is enzymatically active. Western blot analysis of urinary ACE2 demonstrated two prominent immunoreactive bands at approximately 70 & 90 kDa. The predominant immunoreactive band is approximately 20 kDa shorter than the one demonstrated for kidney lysate, indicating possible ectodomain shedding of active renal ACE2 in the urine. Therefore, it is tempting to speculate that renoprotection of rosiglitazone could be partially mediated via downregulation of renal ADAM17 and ACE2 shedding. In addition, there was a positive correlation between blood glucose, urinary albumin, plasma glucagon, and triglyceride levels with urinary ACE2 excretion. In conclusion, urinary ACE2 could be used as a sensitive biomarker of diabetic nephropathy and for monitoring the effectiveness of renoprotective medication.

Effects of enalapril on the expression of cardiac angiotensin-converting enzyme and angiotensin-converting enzyme 2 in spontaneously hypertensive rats

The discovery of angiotensin-converting enzyme 2 (ACE2) has greatly modified understanding of the renin-angiotensin system (RAS). To investigate the cardiac expression of ACE2 and ACE in spontaneously hypertensive rats (SHRs) and the effects of enalapril on them. Fifteen SHRs were randomly assigned to two groups: an SHR control group (n=7), treated with vehicle; and an enalapril group (n=8), treated with enalapril (15 mg/kg/day). After 4 weeks of treatment, the rats were killed and the left ventricular tissue was dissected. Reverse transcription-polymerase chain reaction and Western blot protein staining were performed to detect expression of ACE2 and ACE messenger ribonucleic acid (mRNA) and protein. Ten Wistar Kyoto rats (WKYs) served as the normotensive control group, which were treated with vehicle. Compared with in normotensive WKYs, cardiac expression of ACE mRNA and protein in SHRs was increased (1.68±0.34 vs. 0.33±0.12, P<0.05 and 1.21±0.14 vs. 0.71±0.11, P<0.05, respectively), whereas cardiac expression of ACE2 mRNA and protein was decreased (0.50±0.15 vs. 1.16±0.24, P<0.05 and 0.71±0.24 vs. 1.22±0.14, P<0.05, respectively). After treatment with enalapril, the levels of ACE mRNA and protein were decreased (0.44±0.19 vs. 1.68±0.34, P<0.01 and 0.87±0.13 vs. 1.21±0.14, P<0.05, respectively), the level of ACE2 mRNA was increased (1.77±0.49 vs. 0.50±0.15, P<0.05) but the level of ACE2 protein remained unchanged. In SHRs, the expression of cardiac ACE was remarkably increased, whereas ACE2 was notably decreased. Reduction of ACE and elevation of ACE2 might be one of the mechanisms underlying the antihypertensive function of enalapril.

Living high training low induces physiological cardiac hypertrophy accompanied by down-regulation and redistribution of the renin-angiotensin system.

Living high training low" (LHTL) is an exercise-training protocol that refers living in hypoxia stress and training at normal level of O2. In this study, we investigated whether LHTL caused physiological heart hypertrophy accompanied by changes of biomarkers in renin-angiotensin system in rats. Adult male SD rats were randomly assigned into 4 groups, and trained on living low-sedentary (LLS, control), living low-training low (LLTL), living high-sedentary (LHS) and living high-training low (LHTL) protocols, respectively, for 4 weeks. Hematological parameters, hemodynamic measurement, heart hypertrophy and plasma angiotensin II (Ang II) level of the rats were measured. The gene and protein expression of angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II receptor I (AT1) in heart tissue was assessed using RT-PCR and immunohistochemistry, respectively. LLTL, LHS and LHTL significantly improved cardiac function, increased hemoglobin concentration and RBC. At the molecular level, LLTL, LHS and LHTL significantly decreased the expression of ACE, AGT and AT1 genes, but increased the expression of ACE and AT1 proteins in heart tissue. Moreover, ACE and AT1 protein expression was significantly increased in the endocardium, but unchanged in the epicardium. LHTL training protocol suppresses ACE, AGT and AT1 gene expression in heart tissue, but increases ACE and AT1 protein expression specifically in the endocardium, suggesting that the physiological heart hypertrophy induced by LHTL is regulated by region-specific expression of renin-angiotensin system components.

Association of all-trans retinoic acid treatment with the renin-angiotensin aldosterone system expression in glomerulosclerosis rats

All-trans retinoic acid (ATRA), a promising therapeutic agent, has been confirmed in animal experiments as playing a protective role against renal diseases. The renin-angiotensin aldosterone system (RAAS) plays a key role in the pathogenesis of renal diseases, and RAAS inhibitors can prevent the progression of kidney diseases. In our previous study, we found that ATRA could play a protective role against glomerulosclerosis (GS) lesions in rats, and its effect was similar to RAAS inhibitors. However, whether ATRA treatment was associated with RAAS expression was not clear. Six-week-old male Wistar rats were divided into three groups: sham operation group (SHO), glomerulosclerosis model group without treatment (GS) and GS model group treated with ATRA (GA). At the end of 13 weeks, the relevant samples were collected and analyzed. The mRNA and protein expression of angiotensin-converting enzyme 1 (ACE1) in the GS group was notably higher when compared with the SHO group. However, mRNA and protein expression of ACE1 in the ATRA treatment group was markedly down-regulated when compared with the GS group. Angiotensin-converting enzyme 2 (ACE2) expression (mRNA or protein) in the GS group was reduced compared with that in the SHO group, and ATRA markedly increased the mRNA and protein expression of ACE2 compared with the GS group. The levels of protein expression of angiotensin I and angiotensin II were significantly up-regulated in the GS group compared with those in the SHO group, and ATRA reduced their expression in the GA group when compared with the GS group. ATRA is associated with RAAS expression in GS rats, but its detailed mechanism needs to be elucidated by further research.

Abstract 282: Overexpression of Angiotensin-converting Enzyme 2 Attenuates Tonically Acting Glutamatergic Inputs to the Rostral Ventrolateral Medulla in Hypertensive Rats

The rostral ventrolateral medulla (RVLM) plays a critical role in control of cardiovascular regulation. The increased tonic glutamatergic input to the RVLM contributes to high blood pressure and enhanced sympathetic outflow in hypertensive rats. Recently, it has been reported that overexpression of angiotensin-converting enzyme 2 (ACE2) is capable of lowering high blood pressure in the spontaneously hypertensive rat (SHR). Thus, the aim of this study was to determine the effect of ACE2 gene transfer on tonically acting glutamatergic input to the RVLM in the SHR. It was confirmed that protein expression of ACE2 in the RVLM was increased by an average of 66% in the SHR treated with lentiviral-mediated overexpression of ACE2 (lenti-ACE2) compared with lenti-green fluorescent protein (GFP). A significant reduction in resting blood pressure and the 24-h urinary excretion of norepinephrine was observed in the SHR treated with lenti-ACE2 in the RVLM compared with lenti-GFP. Overexpression of ACE2 significantly reduced the concentration of glutamate (1586±142 μg/L in lenti-GFP versus 621±60 μg/L in lenti-ACE2) from microdialysis-liquid from the RVLM of the SHR. Furthermore, a decrease in resting blood pressure, heart rate, and renal sympathetic nerve activity evoked by microinjection of the glutamate receptor antagonist kynurenic acid (2.7 nmol) into the RVLM was significantly blunted in the SHR treated with lenti-ACE2 compared with lenti-GFP. Based on these data, we suggest that the beneficial effect of ACE2 overexpression in the RVLM on hypertension is associated with reduction in the tonically acting glutamatergic input to the RVLM. (Supported by NSCF: 81170240)

Abstract 374: Genetic Disruption of Guanylyl Cyclase/Natriuretic Peptide Receptor-AUpregulates Renal (Pro)Renin Receptor Gene Expression in Npr1 Mutant Mice

Mice carrying targeted-disruption of guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene ( Npr1 ) exhibits hypertension, cardiac, and kidney hypertrophy, and congestive heart failure. The objective of the present study was to determine the role of (pro)renin receptor (P)RR in the kidneys of Npr1 gene-disrupted (-/-; 0-copy), and wild-type (+/+; 2-copy) mice. Disruption of Npr1 gene leads to increased systolic blood pressure by 36mmHg in 0-copy mice (138.4 ± 3.6) as compared with wild-type mice (102.2 ± 3.2 mm Hg). The renal (P)RR protein and mRNA levels were increased by 3-fold (4.8 ± 0.7 vs.1.3±0.4) and 3.5-fold (5.0 ± 0.5 vs.1.9 ± 0.3), respectively, in 0-copy mice compared with 2-copy control mice. To identify the Angiotensin II-independent mechanism of (P)RR, angiotensin converting enzyme (ACE) inhibitor, captopril and angiotensin II receptor-1 (AT1R) antagonist, losartan were administered to all Npr1 mice genotypes. The protein expression of ACE (3.9 ± 0.6 vs.1.9 ± 0.4; 2-fold) and AT1R (4.5 ± 0.4 vs.1.7; 2.5-fold) were decreased in 0-copy treated mice compared with 0-copy control mice, whereas the (P)RR expression was not altered by both inhibitors. Further, expression of phospho-extracellular regulated kinases (p-Erk1/2, 5.3 ± 0.7 vs 1.6 ± 0.5; 3-fold) and phospho-p38 (p-p38, 8.0 ± 0.8 vs. 2.1 ± 0.7; 4-fold) were increased in 0-copy mice as compared with 2-copy control mice. Treatment with Erk1/2 inhibitor U0126 and p38 inhibitor SB203580 attenuated the levels of p-Erk1/2 (6.8 ± 0.5 ± vs.1.1 ± 0.3; 6-fold) and p-p38 (4.7 ± 0.4 vs. 1.6 ± 0.5; 3-fold) in 0-copy treated mice as compared with 0-copy control mice. Captopril and losartan did not alter the expression levels of p-Erk1/2 and p-p38 in 0-copy mice as compared with 0-copy treated mice. The present results suggests that absence of NPRA/cGMP signaling causes increased expression of (P)RR and leads to MAPKs activation in the Npr1 null mutant mice.