Expression Of Renin-angiotensin System Components Research Articles

Effects of ACE2 deficiency on physical performance and physiological adaptations of cardiac and skeletal muscle to exercise.

The renin-angiotensin system (RAS) is related to physiological adaptations induced by exercise. Angiotensin-converting enzyme (ACE) 2 is a major regulator of the RAS in tissues, as it metabolizes angiotensin (Ang) II to Ang-(1-7). The aim of this study was to determine the effects of ACE2 deficiency on physical performance and physiological adaptations induced by voluntary running. Physical performance, body composition and plasma angiotensin levels, as well as tissue morphology and gene expression of RAS components in the left ventricle (LV) and skeletal muscle (gastrocnemius), were evaluated in ACE2-deficient (ACE2(-/y)) and wild-type (ACE2(+/y)) mice after 6 weeks of voluntary wheel running. ACE2(-/y) mice run less than ACE2(+/y) mice (19±4.7 vs. 26±12.6 revolutions per day × 100, P<0.01). The ACE2(+/y) group presented a lower fat mass (15±1.1%) and higher muscle mass (76.6±1.6%) after 6 weeks of voluntary running compared with the sedentary control group (fat mass: 18.3±2.1%; muscle mass: 72.7±2.2). However, no change in body composition was observed in ACE2(-/y) mice after exercise. Heart and skeletal muscle hypertrophy was observed only in trained ACE2(+/y) mice. Besides a small decrease in Ang I in ACE2(-/y) mice, plasma levels of angiotensin peptides remained unchanged by exercise or ACE2 deficiency. In the LV of trained animals, AT2 gene expression was higher in ACE2(+/y) compared with ACE2(-/y) mice. ACE2 deficiency leads to an increase in AT1 gene expression in skeletal muscle. ACE expression in soleus was increased in all exercised groups. ACE2 deficiency affects physical performance and impairs cardiac and skeletal muscle adaptations to exercise.

ERK1/2 MAPK signaling in hypothalamic paraventricular nucleus contributes to sympathetic excitation in rats with heart failure after myocardial infarction.

Brain MAPK signaling pathways are activated in heart failure (HF) induced by myocardial infarction and contribute to augmented sympathetic nerve activity. We tested whether decreasing ERK1/2 (also known as p44/42 MAPK) signaling in the hypothalamic paraventricular nucleus (PVN), a forebrain source of presympathetic neurons, would reduce the upregulation of sympathoexcitatory mediators in the PVN and augmented sympathetic nerve activity in rats with HF. Sprague-Dawley rats underwent left anterior descending coronary artery ligation to induce HF, with left ventricular dysfunction confirmed by echocardiography. One week after coronary artery ligation or sham operation, small interfering (si)RNAs targeting ERK1/2 or a nontargeting control siRNA was microinjected bilaterally into the PVN. Experiments were conducted 5-7 days later. Confocal images revealed reduced phosphorylated ERK1/2 immunofluorescence in the PVN of HF rats treated with ERK1/2 siRNAs compared with HF rats treated with control siRNA. Western blot analysis confirmed significant reductions in both total and phosphorylated ERK1/2 in the PVN of HF rats treated with ERK1/2 siRNAs along with reduced expression of renin-angiotensin system components and inflammatory mediators. HF rats treated with ERK1/2 siRNAs also had reduced PVN neuronal excitation (fewer Fos-related antigen-like-immunoreactive neurons), lower plasma norepinephrine levels, and improved peripheral manifestations of HF compared with HF rats treated with control siRNAs. These results demonstrate that ERK1/2 signaling in the PVN plays a pivotal role in mediating sympathetic drive in HF induced by myocardial infarction and may be a novel target for therapeutic intervention.

Abstract P115: Effects of Exercise Training on Renal Damage and Renin-angiotensin System in Dahl Salt-sensitive Rats

Exercise training (Ex) has anti-hypertensive and renal protective effects. Renin-angiotensin system (RAS) are involved in the regulation of blood pressure and renal damage. In this study, we investigated the effects of the Ex on renal RAS in Dahl salt-sensitive rats. Six-week-old, male Dahl salt-sensitive rats were divided into four groups: 1) normal salt diet (NS); 2) NS + Ex; 3) high salt diet (HS); 4) HS+ Ex. NS or HS groups were fed diet containing 0.6% or 8% NaCl, and treadmill running was performed in Ex groups for 8 weeks (5 days/week; 60 min/day at 16-20 m/min, 0% grade). Systolic blood pressure (SBP) was monitored by tail-cuff method. Urine samples were collected on ice for 24 hours with metabolic cage. After 8 weeks, protein expression of RAS components in renal cortex (CO) and outer medulla (OM) were investigated by Western blotting. HS significantly elevated SBP (209±6 vs. 114±3 mmHg), and Ex did not change SBP (205±8 mmHg). HS significantly decreased creatinine clearance (0.96±0.04 vs. 2.57±0.07 ml/min/g kidney weight), but Ex significantly mitigated creatinine clearance (1.35±0.18 ml/min/g kidney weight). HS significantly increased urinary protein excretion (433±37 vs. 16±2 mg/day), but Ex significantly suppressed urinary protein excretion (327±22 mg/day). HS induced glomerular sclerosis, but Ex suppressed it. HS increased angiotensinogen expression (138 % and 328 %) and decreased renin expression (47% and 24%) in the CO and OM. HS increased angiotensin II type 1 (AT1) receptor expression in the OM (149%) and Mas receptor expression in the CO (198%), but decreased angiotensin II type 2 (AT2) receptor expression in the CO and OM (53% and 36%) and Mas receptor expression in the OM (20%). Ex improved HS-increased angiotensinogen and AT1 receptor expressions only in the OM. Ex improved HS-decreased renin expression in the CO and OM, and HS-decreased AT2 and Mas receptor expressions only in the OM. These results indicated that Ex improves HS-induced renal damage independently of SBP with specific changes of RAS components in the OM. Ex may have beneficial effects in HS-induced renal damage.

Maternal hyperthyroidism increases the susceptibility of rat adult offspring to cardiovascular disorders

Suboptimal intrauterine conditions as changed hormone levels during critical periods of the development are considered an insult and implicate in physiological adaptations which may result in pathological outcomes in later life. This study evaluated the effect of maternal hyperthyroidism (hyper) on cardiac function in adult offspring and the possible involvement of cardiac Renin-Angiotensin System (RAS) in this process. Wistar dams received orally thyroxin (12 mg/L) from gestational day 9 (GD9) until GD18. Adult offspring at postnatal day 90 (PND90) from hyper dams presented increased SBP evaluated by plethysmography and worse recovery after ischemia-reperfusion (I/R), as evidenced by decreased LVDP, +dP/dT and –dP/dT at 25 min of reperfusion and by increased infarct size. Increased cardiac Angiotensin I/II levels and AT1R in hyper offspring were verified. Herein, we provide evidences that maternal hyperthyroidism leads to altered expression of RAS components in adult offspring, which may be correlated with worse recovery of the cardiac performance after ischemic insults and hypertension.

Perinatally administered losartan augments renal ACE2 expression but not cardiac or renal Mas receptor in spontaneously hypertensive rats.

Since the identification of the alternative angiotensin converting enzyme (ACE)2/Ang-(1-7)/Mas receptor axis, renin-angiotensin system (RAS) is a new complex target for a pharmacological intervention. We investigated the expression of RAS components in the heart and kidney during the development of hypertension and its perinatal treatment with losartan in young spontaneously hypertensive rats (SHR). Expressions of RAS genes were studied by the RT-PCR in the left ventricle and kidney of rats: normotensive Wistar, untreated SHR, SHR treated with losartan since perinatal period until week 9 of age (20 mg/kg/day) and SHR treated with losartan only until week 4 of age and discontinued until week 9. In the hypertrophied left ventricle of SHR, cardiac expressions of Ace and Mas were decreased while those of AT1 receptor (Agtr1a) and Ace2 were unchanged. Continuous losartan administration reduced LV weight (0.43 ± 0.02; P < 0.05 versus SHR) but did not influence altered cardiac RAS expression. Increased blood pressure in SHR (149 ± 2 in SHR versus 109 ± 2 mmHg in Wistar; P < 0.05) was associated with a lower renal expressions of renin, Agtr1a and Mas and with an increase in ACE2. Continuous losartan administration lowered blood pressure to control levels (105 ± 3 mmHg; P < 0.05 versus SHR), however, only renal renin and ACE2 were significantly up-regulated (for both P < 0.05 versus SHR). Conclusively, prevention of hypertension and LV hypertrophy development by losartan was unrelated to cardiac or renal expression of Mas. Increased renal Ace2, and its further increase by losartan suggests the influence of locally generated Ang-(1-7) in organ response to the developing hypertension in SHRs.

Contribution of the renin–angiotensin system in chronic foot-shock induced hypertension in rats

AimsChronic foot shock has been demonstrated to induce hypertension. The present study was designed to explore whether the renin–angiotensin system (RAS) plays a role in this process and the possible mechanisms involved in chronic-foot-shock-induced hypertension. Main methodsMale Sprague–Dawley rats were subjected to a two-week foot shock with or without an angiotensin II (Ang II) type 1 receptor blocker (ARB, candesartan) or an angiotensin I converting enzyme inhibitor (ACEI, captopril). The expression of RAS components in the central nervous and circulatory systems was examined. Antioxidant levels in the plasma were monitored. Key findingsTwo-week foot shock significantly increased systolic blood pressure (SBP). Angiotensinogen, angiotensin I converting enzyme (ACE)-1, ACE-2, angiotensin type 1a and type 1b receptors, and vasopressin (VAP) mRNA expression in the cerebral cortex and hypothalamus were increased along with the concentration of renin and Ang II in the plasma; these changes were accompanied by decreased glutathione peroxidase activity and increased lipid peroxidation levels and plasma corticosterone concentrations. Both candesartan and captopril suppressed not only the increases in SBP but also the increases in VAP expression in the hypothalamus and RAS components in the central nervous system and the circulatory system. The decreases in antioxidant levels and the increases in lipid peroxidation and corticosterone levels were also partially reversed by candesartan or captopril treatment. SignificanceChronic foot shock increases expression of the main RAS components, which play an important role in the development of high blood pressure through increased VAP levels, oxidative stress levels and stress hormone levels.

Involvement of the receptor-associated prorenin system in the pathogenesis of human conjunctival lymphoma.

Extranodal marginal zone B-cell lymphoma (EMZL) is the most common subtype of conjunctival lymphoma, though its molecular mechanisms of pathogenesis are largely unknown. We attempted to explore the association of the renin-angiotensin system (RAS) and (pro)renin receptor ([P]RR) in the pathogenesis of conjunctival lymphoma. Surgically removed conjunctiva EMZL samples were used for gene expression, and immunohistochemical and immunofluorescence analyses of (P)RR and RAS components. Human B-lymphoblast IM-9 cells were treated with prorenin or angiotensin II (Ang II), and gene expression levels were analyzed using real-time quantitative PCR (qPCR). In addition, immunofluorescence analysis of EMZL samples was used to evaluate the in vivo expression of those components. Gene expression and immunohistochemical analyses revealed the expression of RAS components, including (P)RR and angiotensin II type 1 receptor (AT1R), in EMZL tissues. Double-labeling analyses demonstrated that (P)RR and AT1R were detected in cells positive for CD20, a marker for B-cells, where they colocalized with prorenin and angiotensinogen, respectively. Prorenin stimulation of human B-lymphoblast IM-9 cells increased mRNA expression levels of fibroblast growth factor 2 (FGF2), while angiotensin II treatment upregulated the expression levels of basigin (BSG), matrix metallopeptidase (MMP)2, 9, and 14, which were abolished by (P)RR and AT1R blockades, respectively. Immunofluorescence analyses of clinical samples showed colocalizations of (P)RR and AT1R with the products of these upregulated genes. The present study suggests that activation of (P)RR and AT1R is associated with the pathogenesis of conjunctival EMZL by stimulating the production of FGF2 and MMPs.

Expression of classical components of the renin-angiotensin system in the human eye.

The purpose of this study was to determine the relative expression of clinically-relevant components of the renin-angiotensin system (RAS) in the adult human eye. We obtained 14 post-mortem enucleated human eyes from patients whom had no history of inflammatory ocular disease nor pre-mortem ocular infection. We determined the gene expression for prorenin, renin, prorenin receptor, angiotensin-converting enzyme, angiotensinogen and angiotensin II Type 1 receptor, on tissue sections and in cultured human primary retinal pigment epithelial and iris pigment epithelial (RPE/IPE) cell lines, using both qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR). Protein expression was studied using indirect immunofluorescence (IF). Almost all components of the classical RAS were found at high levels, at both the transcript and protein level, in the eyes' uvea and retina; and at lower levels in the cornea, conjunctiva and sclera. There was a much lower level of expression in the reference cultured RPE/IPE cells lines. This study describes the distribution of RAS in the normal adult human eye and demonstrates the existence of an independent ocular RAS, with uveal and retinal tissues showing the highest expression of RAS components. These preliminary findings provide scope for examination of additional components of this system in the human eye, as well as possible differential expression under pathological conditions.

Abstract 4955: Role of Angiotensin II in prostate cancer metastasis

Abstract Introduction: At the initial stages prostate cancer development depends on a crucial level of androgenic stimulation but at later stages when it becomes androgen independent the perturbation from androgen receptor axis leads to the development of more aggressive phenotypes with greater metastatic potential. At this stage the growth and progression of prostate cancer is regulated by various local growth factors and neuro-hormones which are altered in prostate cancer microenvironment. Therefore, identifying these factors which are aberrantly expressed in the tumor microenvironment and understanding how these factors are involved in regulating the tumor progression are essential for developing better targeted therapeutic strategy for the treatment of advanced stages of prostate cancer. Objective: As emerging reports suggest the importance of tissue based renin angiotensin system (RAS) in pathophysiology of the prostate and as studies indicate a lower incidence of cancer and cancer related deaths in patients taking angiotensin receptor blockers (ARBs) (as medication for hypertension), we intend to study the role played by this system in the progression of prostate cancer. Methods and Results: Our results show that there is limited expression of RAS components, Angiotensin II (AT II), the main bioactive effector peptide of RAS and its receptors, and its receptor Angiotensin receptor type 1( AT1R) and Angiotensin receptor type 2 (AT2R) , in mouse prostate tissues. However, RAS becomes activated in prostate cancer. The expressions of ATII and AT1R are significantly higher in androgen independent orthotopic PC3 prostate tumor tissues. The expression of AT2R remains unaltered during the development of tumor. Therefore to evaluate the role of ATII in prostate cancer progression, in vitro experiments using two androgen independent orthotopic cell lines, PC3 and C4-2 were performed. Both these cell lines express AT1R. Our results indicate that ATII acting through AT1R promotes metastatic behavior such as invasion and migration of prostate cancer cells and inhibition of ATII by blocking AT1R receptor abrogates this action of ATII. Conclusions: This study identifies a new role of local RAS in the metastatic progression of prostate cancer and will eventually help to develop newer and effective therapeutic strategies in treatment of androgen independent prostate cancer by targeting ATII or AT1R. This strategy can be used either alone or in combination with existing therapeutic strategies in treatment of the disease. Citation Format: Debanjan Chakroborty, Chandrani Sarkar. Role of Angiotensin II in prostate cancer metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4955. doi:10.1158/1538-7445.AM2014-4955

Nuclear expression of renin-angiotensin system components in NRK-52E renal epithelial cells.

Isolated nuclei of sheep proximal tubules express angiotensin (Ang) receptors as well as angiotensinogen (AGT) and renin. The present study characterized the NRK-52E tubular epithelial cell line for the intracellular expression of renin-angiotensin system (RAS) components. RAS components were visualized by immunofluorescent staining in intact cells and protein expression in isolated nuclei. An antibody to the angiotensin I (Ang I) sequence of AGT (AI-AGT) revealed only cytosolic staining, while an antibody to an internal sequence of AGT (Int-AGT) revealed primarily nuclear staining. Immunoblots of nuclear and cytosolic fractions confirmed the differential cell staining of AGT. Immunostaining for renin was present on nuclei of intact cells. Nuclear renin activity averaged 0.77±0.05 nmol/mg protein/h that was reduced by aliskiren (0.13±0.01 nmol/mg/h, n=3, p<0.01); trypsin activation increased activity three-fold. Peptide staining localized angiotensin II (Ang II) and Ang-(1-7) to the nucleus and peptide content averaged 59±2 and 57±22 fmol/mg (n=4), respectively. Peptide metabolism in isolated nuclei revealed the processing of Ang I to Ang-(1-7) by thimet oligopeptidase. We conclude that the NRK-52E cells express an intracellular RAS localized to the nucleus and may be an appropriate cell model to elucidate the functional relevance of this system.

Abstract 557: ACE2/Angiotensin-(1-7) Stimulates Vasoprotection-Relevant Functions of Human CD34 + Cells

Angiotensin converting enzyme 2 (ACE2) and angiotensin (Ang)-(1-7), key members of the vasoprotective axis of the renin-angiotensin system (RAS), have been shown to modulate hematopoietic functions of bone marrow-derived cells. This coupled with recent studies implicating the involvement of bone marrow stem/progenitor cells in the cardiovascular functions, we evaluated the expression of RAS components and functions of ACE2 and Ang-(1-7) in human CD34 + cells, hematopoietic stem cells with vasoprotective functions. Peripheral blood was obtained from healthy individuals and CD34 + cells were isolated by immunomagnetic separation. Real-time PCR was carried out, and the effects of Ang II, Ang-(1-7), NorLeu 3 Ang-(1-7), and ACE2 activators, diminazene aceturate (DIZI) and a xanthenone derivative (XNT), were evaluated on migration, proliferation and adhesion of CD34 + cells. Paracrine factors produced by CD34 + cells were analyzed. CD34 + cells showed the presence of mRNA transcripts of ACE, ACE2 and Mas1 receptor. In contrast, renin, angiotensinogen, and AT1 and AT2 receptors were not detectable. Treatment with 100nM Ang-(1-7) or 10nM NorLeu 3 Ang-(1-7) stimulated proliferation and migration of CD34 + cells while adhesion of these cells to fibronectin was not affected. Ang-(1-7) treatment altered the paracrine profile of CD34 + cells: increased IL10, IL8, G-CSF, GM-CSF, IGFBP-3 and decorin (P&lt;0.03), and decreased IGF1, thrombopoietin and TGFβ1 levels (P&lt;0.05, n=5). In contrast, 100nM Ang II has no effects on proliferation and migration of CD34 + cells. However, in the presence of Ang II, 100nM DIZI or 100nM XNT increased proliferation and migration of CD34 + cells compared to the cells treated with Ang II alone (P&lt;0.05, n=7). Treatment of total leucocytes with Ang II prior to the isolation of CD34 + cells attenuated their migration to stromal-derived factor-1α and proliferation compared to the untreated (P&lt;0.05, n=5), and enhanced their adhesion to fibronectin. These studies suggest that activation of endogenous ACE2 or treatment with Ang-(1-7) stimulates the functions of CD34 + cells that are indicators of their cardiovascular protective functions while Ang II may attenuate these properties indirectly via acting on leucocytes.

Long-Term Effects of Maternal Diabetes on Blood Pressure and Renal Function in Rat Male Offspring

Aims/HypothesisGestational diabetes mellitus (GDM) is increasing rapidly worldwide. Previous animal models were established to study consequences of offspring after exposure to severe intrauterine hyperglycemia. In this study we are aiming to characterize the blood pressure levels and renal function of male offspring obtained from diabetic mothers with moderate hyperglycemia.MethodsWe established a rat model with moderate hyperglycemia after pregnancy by a single intraperitoneal injection of streptozotocin (STZ). The male offspring were studied and fed with either normal diet or high salt diet after weaning. Arterial pressure and renal function were measured.ResultsArterial pressure of male offspring increased from 12 weeks by exposure to intrauterine moderate hyperglycemia. At 20 weeks, high salt diet accelerated the blood pressure on diabetic offspring compared to diabetic offspring fed with normal diet. We found offspring exposed to intrauterine moderate hyperglycemia had a trend to have a higher creatinine clearance rate and significant increase of urinary N-acetyl-β-D-glucosaminidase (NAG) excretion indicating an early stage of nephropathy progression.Conclusions/InterpretationWe observed the high blood pressure level and early renal dysfunction of male offspring obtained from diabetic mothers with moderate hyperglycemia. Furthermore, we investigated high salt diet after weaning on offspring exposed to intrauterine hyperglycemia could exacerbate the blood pressure and renal function. Renin angiotensin system (RAS) plays an important role in hypertension pathogenesis and altered gene expression of RAS components in offspring with in utero hyperglycemia exposure may account for the programmed hypertension. Therefore, our study provides evidence “fetal programming” of maternal diabetes is critical for metabolic disease development.

Upregulation of a local renin–angiotensin system in the rat carotid body during chronic intermittent hypoxia

The carotid body (CB) plays an important role in the alteration of cardiorespiratory activity in chronic intermittent hypoxia (IH) associated with sleep-disordered breathing, which may be mediated by local expression of the renin-angiotensin system (RAS). We hypothesized a pathogenic role for IH-induced RAS expression in the CB. The CB expression of RAS components was examined in rats exposed to IH resembling a severe sleep-apnoeic condition for 7 days. In situ hybridization showed an elevated expression of angiotensinogen in the CB glomus cells in the hypoxic group when compared with the normoxic control group. Immunohistochemical studies and Western blot analysis revealed increases in the protein level of both angiotensinogen and angiotensin II type 1 (AT1) receptors in the hypoxic group, which were localized to the glomic clusters containing tyrosine hydroxylase. RT-PCR studies confirmed that levels of the mRNA expression of angiotensinogen, angiotensin-converting enzyme, AT1a and AT2 receptors were significantly increased in the CBs of the hypoxic rats. Functionally, the [Ca(2+)]i response to exogenous angiotensin II was enhanced in fura-2-loaded glomus cells dissociated from hypoxic rats when compared with those of the normoxic control animals. Pretreatment with losartan, but not PD123319, abolished the angiotensin II-induced [Ca(2+)]i response, suggesting an involvement of AT1 receptors. Moreover, daily treatment of the IH group of rats with losartan attenuated the levels of oxidative stress, gp91(phox) expression and macrophage infiltration in the CB. Collectively, the upregulated local RAS expression could play a pathogenic role in the augmented CB activity and local inflammation via AT1 receptor activation during IH conditions in patients with sleep-disordered breathing.

Abstract 440: Activation of the Brain Renin-angiotensin System is Critically Involved in the Hypertension Associated With Bbs

Bardet-Biedl syndrome (BBS) is a human autosomal recessive disorder characterized by a pleiotropic of phenotypes including obesity, renal abnormalities, mental retardation, retinal pigmentary dystrophy, polydactyly and hypogenitalism. Hypertension is also common in BBS patients, except BBS2. Consistent with this, we previously demonstrated that obese Bbs2 knockout mice are normotensive while obese Bbs4 null mice are hypertensive. However, the underlying molecular mechanisms of hypertension in BBS remain poorly defined. Using BBS mice, we tested whether abnormalities in the brain renin-angiotensin system (RAS) may contribute to the increased arterial pressure in BBS. Using real time PCR, we found that mRNA expression of RAS components were differentially altered in Bbs2 -/- and Bbs4 -/- mice. Compared to wild type controls, the hypertensive Bbs4 -/- mice exhibited significantly (P&lt;0.05) elevated expression of renin (160%), angiotensin converting enzyme (ACE, 185%), angiotensin type 1a receptor (AT1aR, 143%) and AT1bR (244%) in the brain. Notably, while the expression of ACE was increased (154%) in the brain of the normotensive Bbs2 -/- mice, the expression of renin, AT1aR and AT1bR were significantly (P&lt;0.05) decreased by 56%, 60% and 36%, respectively, relative to controls. Next, we examined the functional consequences of the elevated brain RAS components in Bbs4 -/- mice. Consistent with our previous findings, radiotelemetric mean arterial pressure was significantly (P&lt;0.05) elevated in the obese Bbs4 -/- mice (114±3 mmHg) relative to wild type controls (101±1 mmHg). Moreover, Bbs4 -/- mice exhibited an exaggerated decrease in arterial pressure in response to intracerebroventricular (ICV) administration of losartan (AT1 receptor antagonist). Indeed, ICV losartan (10 g) decreased mean arterial pressure by 24±7 mmHg in Bbs4 -/- mice vs. 5±10 mmHg in wild type controls (P&lt;0.05). Our data indicate that overactivity of the brain RAS may account for the high blood pressure associated with BBS. This study provides a novel molecular mechanism for the hypertension associated with BBS.

Abstract 548: Reduced Urinary Angiotensinogen Excretion in Preeclampsia

Objective: Preeclampsia (preE) is a hypertensive disorder of pregnancy. We reported the suppression of circulatory renin-angiotensin system (RAS) in a rat model of preE. Urinary angiotensinogen has been considered as an indicator of intrarenal angiotensin status in hypertension. Little is known about the urinary angiotensinogen in preE. This study evaluates the level of urinary excretion of angiotensinogen in preE to assess the RAS status. Methods: Normal pregnant (n=57) and preE (n=32) patients were recruited from Scott &amp; White Hospital and had their blood drawn between 21 to 40 weeks of pregnancy. Criteria for diagnosis of preE included blood pressure &gt;140/90 mm Hg and proteinuria &gt;300 mg of protein/24h. Two groups of rats were used: normal pregnant (n=10) and preE rats (n=10) which were given weekly injections of desoxycorticosterone acetate and 0.9% saline to drink. Urinary angiotensinogen levels and the plasma AngII levels were assayed by ELISA. The kidney expression of (pro)renin receptor, AT 1 receptor, AT 2 receptor, and renin for the two groups of rats was measured by western blot. Results are expressed as means with SD and comparisons made using Student’s t test with p&lt;0.05 considered significant. Results: In preE patients, the mean urinary excretion of angiotensinogen (2.0 ± 1.1 ng/mg creatinine) differed (p&lt;0.05) from that in patients with normal pregnancy (2.7 ± 1.5 ng/mg creatinine). The urinary excretion of angiotensinogen (PreE: 1.5 ± 0.3, NP: 2. 3 ± 0.4 nmol/day) also differed (p&lt;0.05) in preE rats compared to NP. The plasma concentration of Ang II for preE rats (25 ± 3 fmol/ml) differed (p&lt;0.05) compared to NP rats (39 ± 5 fmol/ml). The kidney expression of (pro)renin receptor was downregulated (p=0.008), while the AT 1 receptor was upregulated (p=0.01) in preE rats compared to NP. However, the kidney expression of AT 2 receptor (1.0 vs 1.1) and renin (1.0 vs 1.4) were similar in NP and PDS rats (p=0.63 and p=0.12, respectively). Conclusions: We demonstrated that urinary excretion of angiotensinogen is reduced in both patients with preE and in a rat model of preE. The kidney expression of RAS components was either downregulated or remained unchanged in preE rats. This finding indicates that preE is an Ang-II independent form of systemic hypertension.

Atrial tachyarrhythmia causes atrial imbalance of the Ace/Ace2 ratio in aged and hypertensive rats

Purpose: Arterial hypertension and age are the most predominant factors causing atrial fibrillation (AF). The exact proarrhythmogenic pathways involved are still unknown. The present study was designed to examine changes in the composition of atrial members of the renin-angiotensin system (RAS) in response to tachyarrhythmia in the presence of arterial hypertension at different ages. The local RAS plays a crucial role in remodelling processes in the heart, primarily through the action of angiotensin II (AngII), which is generated by angiotensin converting enzyme (ACE). ACE2 counterbalances the action of ACE by cleaving of AngII into peptides that act protective. Methods: Atrial tissue from spontaneously hypertensive male rats (SHR; n=6) and their normotensive controls, Wistar-Kyoto (n=6), were analysed at the age of 12 weeks and 6-8 months. Atrial slices were rapidly stimulated at 5 Hz up to 20h to resemble AF (n=4 per group and age). The changes in the expression of RAS components (ACE, ACE2, angiotensin receptors) as well as RAS-associated downstream effectors were estimated at the mRNA and protein levels. Moreover, the activation of MAPK and NF-κB signalling were analysed by immunoblotting. Results: Our data revealed that left atrium of SHR expressed constantly high levels of ACE protein (3.8±0.7; p=0.03) and mRNA (1.7±0.7; p=0.04) compared to normotensive controls (1.0) in each age group. ACE2-mRNA levels did not show any changes at 12 weeks between normotensive and hypertensive rats. With age the mRNA levels of ACE2 diminished in SHR (0.79±0.04; p=0.02). These molecular changes were accompanied by activation of MAPK, NF-κB signalling and the presence of the active caspase-3 in SHR. In response to the stimulation in the electrical field, the atrial tissue from hypertensive rats showed 1.6-fold (p=0.01) and of normotensive rats 3.0-fold increase (p=0.01) in the expression of ACE. In normotensive young rats, ACE2 counterbalanced this action at the mRNA (1.5±0.12; p=0.01) and protein level (2.7±0.80; p=0.11). This protective action of ACE2 decreased with age in normotensive rats, and it disappeared completely in SHR (mRNA: 0.94±0.13; protein: 0.98±0.11). Conclusions: Our data show that hypertension in SHR is accompanied by enhanced atrial expression of angiotensin-converting enzyme. This fact strongly implies a sustained exposure of atrial tissue to elevated levels of local AngII. The observed imbalance of the ACE/ACE2 ratio in aged hypertensive rats might facilitate the susceptibility to arrhythmia especially in this clinical scenario.

Angiotensin II Type 1 Receptor Antagonist Attenuates Lacrimal Gland, Lung, and Liver Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2d) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.

Local renin-angiotensin system in the thyroid – myth or fact?

Beside the basic control system associated with the hypothalamus-hypophysis-thyroid axis and thyrotropin, a growing evidence suggests that thyroid function is influenced by a vast spectrum of locally produced factors. Renin-angiotensin system (RAS) is considered as one of the most powerful and effective regulatory systems in humans. In the “classical” cascade of RAS, angiotensinogen is enzymatically conversed to the most active element of RAS – angiotensin (Ang) II. Ang II exerts its effects via two main receptors: AT1R and AT2R. The understanding of RAS biological function was significantly changed by the discovery of new enzymatic cascades leading to the production of molecules with different than Ang II functional properties – for instance Ang (1-7), molecule reacting with its specific receptor – Mas. Beside the growing complexity of RAS-structure, diverse expression patterns of RAS components in various tissues formed a basis for a concept of “local RAS” in many organs including brain and immune system. It was shown that many organ-specific effects of hormonal factors, including the influence of thyroid hormones on cardiovascular system, were dependent on the modulation of local RAS components. However, very little is known about the role of RAS in the control of thyroid function. In our immunohistochemical analysis of operative specimens we observed for the first time expression of RAS components in human thyroid. Interestingly, although our recently published findings suggest a direct regulatory influence of thyroid function on the phenotype and activity of immune cells in humans, no studies regarding the influence of human RAS on the course of such common inflammatory process as thyroiditis are available. Flow Cytometry analyses performed in our laboratory on thyroid fine needle aspiration biopsy material allowed us to assess quantitatively and phenotypically the immune cell populations of local thyroid blood. The comparison of results obtained with paired peripheral blood and local thyroid blood samples suggested differences in the structure of antigen presenting cell populations, e.g. monocytes, known to be the main source of RAS components in the immune system. Interestingly, RAS positive immune cells have been shown to correlate with clinical course of various inflammatory and malignant diseases. In conclusion, better knowledge about local RAS in the thyroid seems to be of great importance for the understanding of both: thyroid physiology and pathological processes associated with inflammatory and malignant disorders of this gland.

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.

Aberrant Activation of the Intrarenal Renin-Angiotensin System in the Developing Kidneys of Type 2 Diabetic Rats

We have previously reported that intrarenal angiotensin II (Ang II) levels are increased long before diabetes becomes apparent in obese Otsuka-Long-Evans-Tokushima-Fatty (OLETF) rats, a model of type 2 diabetes. In this study, we examined the changes in intrarenal renin-angiotensin system (RAS) activity in the developing kidneys of OLETF rats. Ang II contents and mRNA levels of RAS components were measured in male OLETF and control Long-Evans Tokushima (LETO) rats at postnatal days (PND) 1, 5, and 15, and at 4-30 weeks of age. In both LETO and OLETF rats, kidney Ang II levels peaked at PND 1, then decreased during the pre- and post-weaning periods. However, Ang II levels and gene expression of RAS components, including angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE), were not significantly different between LETO and OLETF rats. Intrarenal Ang IIcontents further decreased during puberty (from 7 to 11 weeks of age) in LETO rats, bur not in OLETF rats. At 11 weeks of age, kidney Ang II levels, urinary AGT excretion, and mRNA levels of AGT and renin were higher in OLETF rats than in LETO rats, while blood glucose levels were not significantly different between these groups of rats. These data indicate that continued intrarenal expression of Ang II during pubescence contributes to the increases in intrarenal Ang II levels in prediabetic OLETF rats, and is associated with increased intrarenal AGT and renin expression. Inappropriate activation of the intrarenal RAS in the prediabetic stage may facilitate the onset and development of diabetic nephropathy in later life.