Overexpression Of Renin Research Articles

Renin Inhibition Attenuates Insulin Resistance, Oxidative Stress, and Pancreatic Remodeling in the Transgenic Ren2 Rat

Emerging evidence indicates that pancreatic tissue expresses all components of the renin-angiotensin system. However, the functional role is not well understood. This investigation examined renin inhibition on pancreas structure/function in the transgenic Ren2 rat harboring the mouse renin gene, a model of tissue renin overexpression. Renin is the rate-limiting step in the generation of angiotensin II (Ang II), which stimulates the generation of reactive oxygen species in a variety of tissues. Overexpression of renin in Ren2 rats results in hypertension, insulin resistance, and cardiovascular and renal damage. Young (6-7 wk old) insulin-resistant male Ren2 and age-matched insulin sensitive Sprague Dawley rats were treated with the renin inhibitor, aliskiren (50 mg/kg.d by ip injection), or placebo for 21 d. At 21 d, the Ren2 demonstrated insulin resistance with increased islet insulin, Ang II, and reduced total insulin receptor substrate (IRS)-1, IRS-2, and Akt immunostaining. There was increased islet nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and subunits (p47(phox) and Rac1) as well as increased nitrotyrosine immunostaining (each P < 0.05). These functional abnormalities were associated with a disordered islet architecture; increased islet-exocrine interface, pericapillary fibrosis, and structurally abnormal mitochondria and content in endocrine and exocrine pancreas. In vivo treatment with aliskiren normalized systemic insulin resistance and islet insulin, Ang II, NADPH oxidase activity/subunits, and nitrotyrosine and improved total IRS-1 and Akt phosphorylation (each P < 0.05) as well as islet/exocrine structural abnormalities. Collectively, these data suggest that pancreatic functional/structural changes are driven, in part, by tissue renin-angiotensin system-mediated increases in NADPH oxidase and reactive oxygen species generation, abnormalities attenuated with direct renin inhibition.

Overexpression of renin in the liver impairs glucose tolerance

Animal and clinical studies have pointed to a potential role of the Renin Angiotensin System (RAS) in diabetes. However, mechanisms underlying this association are not completely understood. The objective of this work is to dissect the mechanisms by which overexpression of RAS mediates glucose intolerance, using a transgenic mouse model overexpressing renin in the liver (RenTg; Caron et al., 2002), resulting in constitutively elevated plasma angiotensin II (Ang II). Twenty week-old RenTg mice exhibit significantly impaired glucose tolerance and fasting insulinemia, despite normal glycemia, leptinemia and adiponectinemia, as compared to their wild type littermates. Although adipokine gene expression did not differ across genotypes, the type 2 Ang II receptor was markedly down-regulated in adipose tissue of RenTg mice. Comparative proteomics of adipose tissue from these mice indicates a genotype-dependent differential expression of several proteins associated with diabetes and glucose metabolism. We are currently studying these candidate proteins to identify their role in RAS-induced diabetes. These studies demonstrate that overexpression of renin impairs glucose tolerance and further support the role of RAS in the pathogenesis of diabetes. Future studies will dissect mechanism(s) and tissue(s) linking RAS to diabetes. This work was supported by the TN agricultural experiment station and by the Office of Science, U.S. Dept. of Energy.

Discoordinate regulation of renal nitric oxide synthase isoforms in ovariectomized mRen2.Lewis rats

Estrogen depletion markedly exacerbates hypertension in female congenic mRen2. Lewis rats, a model of tissue renin overexpression. Because estrogen influences nitric oxide synthase (NOS) and NO may exert differential effects on blood pressure, the present study investigated the functional expression of NOS isoforms in the kidney of ovariectomized (OVX) mRen2. Lewis rats. OVX-mRen2. Lewis exhibited an increase in systolic blood pressure (SBP) of 171 +/- 5 vs. 141 +/- 7 mmHg (P < 0.01) for intact littermates. Renal cortical mRNA and protein levels for endothelial NOS (eNOS) were reduced 50-60% (P < 0.05) and negatively correlated with blood pressure. In contrast, cortical neuronal NOS (nNOS) mRNA and protein levels increased 100 to 300% (P < 0.05). In the OVX kidney, nNOS immunostaining was more evident in the macula densa, cortical tubules, and the medullary collecting ducts compared with the intact group. To determine whether the increase in renal nNOS expression constitutes a compensatory response to the reduction in renal eNOS, we treated both intact and OVX mRen2. Lewis rats with the selective nNOS inhibitor L-VNIO from 11 to 15 wk of age. The nNOS inhibitor reduced blood pressure in the OVX group (185 +/- 3 vs. 151 +/- 8 mmHg, P < 0.05), but pressure was not altered in the intact group (146 +/- 4 vs. 151 +/- 4 mmHg). In summary, exacerbation of blood pressure in the OVX mRen2. Lewis rats was associated with the discoordinate regulation of renal NOS isoforms. Estrogen sensitivity in this congenic strain may involve the influence of NO through the regulation of both eNOS and nNOS.

Approaches to Establishing Angiotensin II as a Neurotransmitter Revisited

During the past 3 to 4 decades, there has been debate on the presence as well as the role of local tissue renin–angiotensin systems (RAS) in cardiovascular physiology and pathophysiology. Although it is now accepted that all components of the RAS are present in a variety of extrarenal tissues and their regulation may be independent of the circulating hormonal system, the factors contributing to this regulation are still not well understood. Also apparent is that our understanding of the relationships among various cell types expressing individual RAS components and production of specific angiotensin peptides in each tissue is still lacking. This is particularly true of the brain RAS, where even after 20years the question of whether there are angiotensinergic versus reninergic neurons or a complete functioning RAS in cerebrospinal fluid (CSF) or extracellular fluid or glia remains in question.1 Ever since early reports provided biochemical evidence of RAS components in brain, controversy regarding their cellular localization, independence from the circulating system, and authenticity of the proteins and peptides persists. It is well accepted that angiotensinogen is present in CSF/interstitial fluid and localization via immunocytochemistry and in situ hybridization histochemistry reveals that production of the precursor protein is primarily in glia, but also in neurons, within key cardiovascular nuclei. Lingering questions remain concerning local expression of authentic renin in tissues, especially given that prorenin or active renin can be sequestered from the circulation2 and other enzymes can exhibit similar proteolytic profiles under certain conditions. However, there is unequivocal evidence of discrete cells within the pituitary, choroid plexus, medulla oblongata, and hypothalamus that are positive for renin immunoreactivity colocalizing mainly with neurons, but in the medulla oblongata and subfornical organ, in glial elements as well. Evidence of renin mRNA in brain tissue provides a mechanism for local synthesis of the …

Autonomic control in rats with overactivity of tissue renin–angiotensin or kallikrein–kinin system

The renin–angiotensin system (RAS) plays an important role in the regulation of the cardiovascular system and the kallikrein–kinin system (KKS) appears to counteract most of the RAS effects. In this study the vagal and the sympathetic influences on the heart rate and the baroreflex control of the heart rate were evaluated in transgenics rats with human tissue kallikrein gene expression [TGR(hKLK1)], and transgenics rats with tissue renin overexpression [TGR(mREN2)27]. Heart rate was similar in all groups but mean arterial pressure was higher in mREN2 rats than in kallikrein and control rats (149 ± 4 vs. 114 ± 3 vs. 113 ± 3 mm Hg, respectively). The intrinsic heart rate was lower in mREN2 rats than in kallikrein and control rats (324 ± 5 vs. 331 ± 3 vs. 343 ± 7 bpm). The HR response to atropine was similar but the response to propranolol was higher in kallikrein rats than control group (61 ± 7 vs. 60 ± 9 vs. 38 ± 7 bpm, respectively). The vagal tonus was lower in mREN2 than in SD and hKAL rats (18 ± 3 vs. 40 ± 6 vs. 35 ± 6 bpm) whereas the sympathetic tonus was higher in kallikrein rats (118 ± 7 vs. 96 ± 1 vs. 81 ± 9 bpm in the mREN2 and SD rats), respectively. Baroreflex sensitivity to bradycardic responses was attenuated in mREN2 rats (0.37 ± 0.05 vs. 1.34 ± 0.08 vs. 1.34 ± 0,13 bpm/mm Hg) while the tachycardic responses were unchanged. The bradycardic responses to electrical stimulation of the vagal nerve were depressed in both renin and kallikrein rats (129 ± 47 vs. 129 ± 22 vs. 193 ± 25 bpm in control group in response to 32 Hz). In conclusion: 1.The rats with overexpression of renin showed decreased intrinsic heart rate and impairment of vagal function, characterized by decreased vagal tonus, reduced response of HR to electrical stimulation of vagus nerve, and depressed reflex bradycardia provoked by increases of blood pressure. 2. The rats with overexpression of kallikrein showed an increase of sympathetic activity that regulates the heart rate, characterized by increased HR response to propranolol and increased sympathetic tonus, accompanied by decreased bradycardic responses to electrical vagal stimulation.

Glia- and Neuron-specific Expression of the Renin-Angiotensin System in Brain Alters Blood Pressure, Water Intake, and Salt Preference

The purpose of this study is to examine the regulation of blood pressure and fluid and electrolyte homeostasis in mice overexpressing angiotensin II (Ang-II) in the brain and to determine whether there are significant physiologic differences in Ang-II production in neurons or glia. Therefore, we generated and characterized transgenic mice overexpressing human renin (hREN) under the control of the glial fibrillary acidic protein (GFAP) promoter (GFAP-hREN) and synapsin-I promoter (SYN-hREN) and bred them with mice expressing human angiotensinogen (hAGT) under the control of the same promoters (GFAP-hAGT and SYN-hAGT). Both GFAP-hREN and SYN-hREN mice exhibited the highest hREN mRNA expression in the brain and had undetectable levels of hREN protein in the systemic circulation. In the brain of GFAP-hREN and SYN-hREN mice, hREN protein was observed almost exclusively in astrocytes and neurons, respectively. Transgenic mice overexpressing both hREN and hAGT transgenes in either glia or neurons were moderately hypertensive. In the glia-targeted mice, blood pressure could be corrected by intracerebroventricular injection of the Ang-II type 1 receptor antagonist losartan, and intravenous injection of a ganglion blocking agent, but not an arginine vasopressin V1 receptor antagonist, lowered blood pressure. These data suggest that stimulation of Ang-II type 1 receptors in the brain by Ang-II derived from local synthesis of renin and angiotensinogen can cause an elevation in blood pressure via a mechanism involving enhanced sympathetic outflow. Glia- and neuron-targeted mice also exhibited an increase in drinking volume and salt preference, suggesting that chronic overexpression of renin and angiotensinogen locally in the brain can result in hypertension and alterations in fluid homeostasis.

It’s Renin in the Brain

Since the first description of renin, angiotensinogen and angiotensin as well as converting enzyme and angiotensin receptors in the brain exactly 30 years ago,1–5⇓⇓⇓⇓ there was a heated debate, whether an endogenous intrinsic brain renin- angiotensin system (RAS) exists or not.6,7⇓ For a long time, the “believers” of local angiotensin II generation by the brain RAS were in a minority. Even at a symposium 1981, celebrating the 10-year anniversary of the discovery of the brain RAS, there was skepticism and the question whether the “renin-like” enzyme in brain was identical with cathepsins, tonin, or other proteases was a matter of debate. Historically, it is interesting that the presence in the brain of other classically peripheral peptides such as insulin, glucagon, and substance P was accepted without identification of the local synthetic pathway and less controversial than angiotensin as neuropeptide. Open letters, public debates, and personal accusations nourished sometimes emotional controversy whether angiotensin could be generated locally in tissue. The brain was a model for angiotensin generating pathways in other extrarenal tissues because the blood-brain barrier, which is impermeable for proteins and peptides, made it more unlikely that the RAS components measured in the brain were contaminations from the plasma. Angiotensin has extremely powerful effects on the brain that synergistically increase blood volume and blood pressure, eg, by stimulation of water intake and salt appetite, release of various pituitary hormones, increase of sympathetic tone, and decrease of baroreceptor reflex.7 The local generation of angiotensin II in the brain by an endogenous intrinsic RAS could therefore be extremely important for cardiovascular control and beyond. With the advent of transgenic technology, novel approaches to study the mechanistic and functional aspects of the brain RAS became available. Renin and angiotensinogen could be overexpressed or ablated …

Angiotensin II as a player in fibrosis

It is generally believed that excess angiotensin is Cre–loxP system. Cre is an enzyme which catalyses a involved in the progression of glomerular and tubuloin- site-specific recombination at a unique 34 bp sequence, terstitial injury and vascular hypertrophy. In this called loxP. We separately introduced a mutation regard, the phenotype of the lack of angiotensin containing loxP sites into Ren1 and Ren2 by gene revealed by angiotensinogen gene null-mutant mice targeting, and established a mouse strain carrying each seemed to be quite paradoxical. They showed hypo- mutation. These two mutants and a transgenic mouse trophic papilla, renal vascular hypertrophy, glomerular line ubiquitously overexpressing Cre recombinase were sclerosis, tubular degeneration and interstitial fibrosis, mated to produce the mice carrying the mutant Ren1 in addition to hypotension and overexpression of renin and Ren2 genes, as well as the Cre transgene concur[1]. Null-mutant mice for the genes of AT1, i.e. AT1A rently. Initially, two mutant Ren genes were located and B [2], but not AT2 [3], showed similar phenotypes, on separate chromosomes. Analysis of the renin genoindicating that these abnormal phenotypes are caused type of the offspring revealed that 7/73 (9.6%) had by the lack of angiotensin’s function mediated by AT1 both Ren1 and Ren2 mutation, and that 6/73 (8.2%) receptors. had no mutant gene. Southern analysis confirmed that Although AT1- or angiotensinogen-deficient mice in the mice with the dual mutation, two mutant Rens did not show urinary tract obstruction, the hypotrophic were recombined at the loxP site. Thus we have papilla observed in these mice was very similar to the successfully generated Ren1 and Ren2 dual targeted phenotype of hydronephrosis generated by ureteral mice. obstruction. In AT1-deficient mice, a renal pelvis was In the resultant dual renin allele, the HA and lacZ not developed and the ureteral smooth muscle layer reporter genes, together with the Ren1 promoter, were was hypotrophic and lacked peristaltic movements [4]. incorporated. Immuno- and histochemical staining Angiotensin II induced the ureteral smooth muscles in revealed that HA and lacZ were expressed in reninorgan cultures of wild-type, but not mutant, ureteral producing cells in the kidney of heterozygotes. In tissues. These results indicate that angiotensin II homozygous mice, HA and lacZ expression were has a critical role in ureteral development and that extended into afferent arterioles, interlobular arteries the hypotrophic papilla in AT1-or angiotensinogen- and glomeruli, thereby representing highly activated deficient mice is a consequence of functional ureteral Ren1 promoter activities. No renin staining was obstruction. detected in HA-positive cells in homozygous mice. In both angiotensinogen- and AT1-deficient mice, Reverse transcriptase–polymerase chain reaction the hypertrophied vasculature is accompanied by renin (RT–PCR) amplification revealed that homozygous overproduction. We speculated that renin per se may mice express a small amount of shorter RNA, which act as a growth factor for the renal vasculature, consists of a part of Ren2, Ren1 and the reporter gene. independently of angiotensin generation. To test this Renin activity in the kidney of homozygous mice (6w, possibility, we generated renin knockout mice [5]. n=5) was undetectable, indicating that the renin genes Mouse strains commonly used for gene targeting have are functionally inactivated in homozygous mice. duplicated renin genes, Ren1 and Ren2. These two Blood pressure measured by the direct method is genes, both producing active renin in the kidney, span 85.6±6.6/53.2±6.6 mmHg in conscious homozygous >40 kb in tandem on chromosome 1. This close prox- (6w, n=5)vs 132.7±1.3/93.0±1.4 mmHg in wild-type imity makes the generation of dual gene-targeted mut- mice (6w, n=3). The kidney of homozygous mice ants by mating single gene-targeted mutants nearly showed medial hypertrophy in afferent arterioles and impossible. To solve this problem, we utilized the interlobular arteries, as well as expansion of the mesangium matrix and hypotrophic papilla, in the same

Beta-adrenergic signal transduction following carvedilol treatment in hypertensive cardiac hypertrophy.

Treatment with the beta-blocker carvedilol leads to an improvement of outcome and ejection fraction in heart failure. These effects occur without affecting the number of beta-adrenergic receptors, as determined in right ventricular biopsies from patients with heart failure. This study was aimed at investigating the effects of carvedilol on beta-adrenergic signal transduction alterations in a model of left ventricular pressure overload, which is characterized by sympathetic activation and a desensitized beta-adrenergic signal transduction. Transgenic rats with overexpression of renin [TG(mREN2)27] were treated with carvedilol (30 micrograms/kg) or held under control conditions and were compared with Sprague-Dawley rats. Myocardial beta-adrenoceptors (125I-labeled iodocyanopindolol binding), Gi alpha (pertussis toxin labeling), Gs alpha-activity (reconstitution into cyc--S49 membranes) and adenylyl cyclase activity were measured. Blood pressure and heart rate, increase in heart rate during sacrifice and pressure rate products were determined. beta-Adrenoceptors were downregulated and Gi alpha-protein levels were significantly increased, producing a desensitization of basal, isoprenaline- and guanine nucleotide-stimulated adenylyl cyclase activity compared to controls. Carvedilol reduced heart rate, blood pressure and pressure rate product in TG(mREN2)27. Carvedilol did not restore biochemical alterations, but even further reduced beta-adrenoceptor numbers and adenylyl cyclase. It exhibited a two affinity state, guanine nucleotide-sensitive binding to cardiac beta-adrenergic receptors similar to isoprenaline but different from metoprolol. Carvedilol did not restore beta-adrenergic signal transduction at concentrations producing antiadrenergic effects in vivo. This effect might be due to an atypical guanine nucleotide-dependent interaction with beta-adrenergic receptors. Thus, ancillary properties could explain the recently reported beneficial effects in patients with heart failure independent from an upregulation of beta-adrenergic receptors.