Intracellular Renin Research Articles

ER ribosomal-binding protein 1 regulates blood pressure and potassium homeostasis by modulating intracellular renin trafficking

BackgroundGenome-wide association studies (GWASs) have linked RRBP1 (ribosomal-binding protein 1) genetic variants to atherosclerotic cardiovascular diseases and serum lipoprotein levels. However, how RRBP1 regulates blood pressure is unknown.MethodsTo identify genetic variants associated with blood pressure, we performed a genome-wide linkage analysis with regional fine mapping in the Stanford Asia–Pacific Program for Hypertension and Insulin Resistance (SAPPHIRe) cohort. We further investigated the role of the RRBP1 gene using a transgenic mouse model and a human cell model.ResultsIn the SAPPHIRe cohort, we discovered that genetic variants of the RRBP1 gene were associated with blood pressure variation, which was confirmed by other GWASs for blood pressure. Rrbp1- knockout (KO) mice had lower blood pressure and were more likely to die suddenly from severe hyperkalemia caused by phenotypically hyporeninemic hypoaldosteronism than wild-type controls. The survival of Rrbp1-KO mice significantly decreased under high potassium intake due to lethal hyperkalemia-induced arrhythmia and persistent hypoaldosteronism, which could be rescued by fludrocortisone. An immunohistochemical study revealed renin accumulation in the juxtaglomerular cells of Rrbp1-KO mice. In the RRBP1-knockdown Calu-6 cells, a human renin-producing cell line, transmission electron and confocal microscopy revealed that renin was primarily retained in the endoplasmic reticulum and was unable to efficiently target the Golgi apparatus for secretion.ConclusionsRRBP1 deficiency in mice caused hyporeninemic hypoaldosteronism, resulting in lower blood pressure, severe hyperkalemia, and sudden cardiac death. In juxtaglomerular cells, deficiency of RRBP1 reduced renin intracellular trafficking from ER to Golgi apparatus. RRBP1 is a brand-new regulator of blood pressure and potassium homeostasis discovered in this study.Graphical

Blood Pressure Elevation of Tubular Specific (P)RR Transgenic Mice and Lethal Tubular Degeneration due to Possible Intracellular Interactions between (P)RR and Alternative Renin Products

The prorenin/renin receptor ((P)RR) is a multifunctional protein that is widely distributed in various organs. Despite intensive research for more than 20 years, this receptor has not been fully characterized. In this study, we generated mice overexpressing the tubular epithelial (P)RR gene ((P)RR-TG mice) to test the previously reported functional role of (P)RR by Ramkumar et al. in 2015 using tubular specific (P)RR KO mice. (P)RR-TG mice were maintained and analyzed in individual metabolic cages and were administered angiotensin II blocker (ARB), direct renin inhibitor (DRI), and bafilomycin, that is, vacuolar ATPase (V-ATPase) antagonist. (P)RR-TG mice were hypertensive and had alkalized urine with lower osmolality and Na+ excretion. ARB and DRI, but not bafilomycin, concurrently decreased blood pressure. Bafilomycin acidized urine of (P)RR-TG mice, or equivalently this phenomenon restored the effect of overexpressed transgene, suggesting that (P)RR functioned as a V-ATPase in renal tubules. Afterall, (P)RR-TG mice were mated with alternative renin transgenic mice (ARen2-TG), which we identified as intracellular renin previously, to generate double transgenic mice (DT-TG). Lethal renal tubular damage was observed in DT-TG mice, suggesting that intracellular renin may be a ligand for (P)RR in tubules. In summary, (P)RR did not substantially affect the tissue renin-angiotensin system (RAS) in our model of tubular specific (P)RR gene over-expression, but alternative intracellular renin may be involved in (P)RR signaling in addition to conventional V-ATPase function. Further investigations are warranted.

Low Nitric Oxide Bioavailability Increases Renin Production in the Collecting Duct.

In the kidney, the stimulation of renin production by the collecting duct (CD-renin) contributes to the development of hypertension. The CD is a major nephron segment for the synthesis of nitric oxide (NO), and low NO bioavailability in the renal medulla is associated with hypertension. However, it is unknown whether NO regulates renin production in the CD. To test the hypothesis that low intrarenal NO levels stimulate the production of CD-renin, we first examined renin expression in the distal nephron segments of CD-eNOS deficient mice. In these mice, specific CD-renin immunoreactivity was increased compared to wild-type littermates; however, juxtaglomerular (JG) renin was not altered. To further assess the intracellular mechanisms involved, we then treated M-1 cells with either 1 mM L-NAME (L-arginine analog), an inhibitor of NO synthase activity, or 1 mM NONOate, a NO donor. Both treatments increased intracellular renin protein levels in M-1 cells. However, only the inhibition of NOS with L-NAME stimulated renin synthesis and secretion as reflected by the increase in Ren1C transcript and renin protein levels in the extracellular media, respectively. In addition, NONOate induced a fast mobilization of cGMP and intracellular renin accumulation. These response was partially prevented by guanylyl cyclase inhibition with ODQ (1H-[1,2,4] oxadiazolo[4,3-a]quinoxalin-1]. Accumulation of intracellular renin was blocked by protein kinase G (PKG) and protein kinase C (PKC) inhibitors. Our data indicate that low NO bioavailability increases CD-renin synthesis and secretion, which may contribute to the activation of intrarenal renin angiotensin system.

Intratubular and intracellular renin-angiotensin system in the kidney: a unifying perspective in blood pressure control.

The renin-angiotensin system (RAS) is widely recognized as one of the most important vasoactive hormonal systems in the physiological regulation of blood pressure and the development of hypertension. This recognition is derived from, and supported by, extensive molecular, cellular, genetic, and pharmacological studies on the circulating (tissue-to-tissue), paracrine (cell-to-cell), and intracrine (intracellular, mitochondrial, nuclear) RAS during last several decades. Now, it is widely accepted that circulating and local RAS may act independently or interactively, to regulate sympathetic activity, systemic and renal hemodynamics, body salt and fluid balance, and blood pressure homeostasis. However, there remains continuous debate with respect to the specific sources of intratubular and intracellular RAS in the kidney and other tissues, the relative contributions of the circulating RAS to intratubular and intracellular RAS, and the roles of intratubular compared with intracellular RAS to the normal control of blood pressure or the development of angiotensin II (ANG II)-dependent hypertension. Based on a lecture given at the recent XI International Symposium on Vasoactive Peptides held in Horizonte, Brazil, this article reviews recent studies using mouse models with global, kidney- or proximal tubule-specific overexpression (knockin) or deletion (knockout) of components of the RAS or its receptors. Although much knowledge has been gained from cell- and tissue-specific transgenic or knockout models, a unifying and integrative approach is now required to better understand how the circulating and local intratubular/intracellular RAS act independently, or with other vasoactive systems, to regulate blood pressure, cardiovascular and kidney function.

Angiotensin generation in the brain: a re-evaluation.

The existence of a so-called brain renin-angiotensin system (RAS) is controversial. Given the presence of the blood-brain barrier, angiotensin generation in the brain, if occurring, should depend on local synthesis of renin and angiotensinogen. Yet, although initially brain-selective expression of intracellular renin was reported, data in intracellular renin knockout animals argue against a role for this renin in angiotensin generation. Moreover, renin levels in brain tissue at most represented renin in trapped blood. Additionally, in neurogenic hypertension brain prorenin up-regulation has been claimed, which would generate angiotensin following its binding to the (pro)renin receptor. However, recent studies reported no evidence for prorenin expression in the brain, nor for its selective up-regulation in neurogenic hypertension, and the (pro)renin receptor rather displays RAS-unrelated functions. Finally, although angiotensinogen mRNA is detectable in the brain, brain angiotensinogen protein levels are low, and even these low levels might be an overestimation due to assay artefacts. Taken together, independent angiotensin generation in the brain is unlikely. Indeed, brain angiotensin levels are extremely low, with angiotensin (Ang) I levels corresponding to the small amounts of Ang I in trapped blood plasma, and Ang II levels at most representing Ang II bound to (vascular) brain Ang II type 1 receptors. This review concludes with a unifying concept proposing the blood origin of angiotensin in the brain, possibly resulting in increased levels following blood-brain barrier disruption (e.g. due to hypertension), and suggesting that interfering with either intracellular renin or the (pro)renin receptor has consequences in an RAS-independent manner.

Establishing an in vitro model of olfactory receptor 78‐mediated renin release

Recent studies by our laboratory have identified Olfactory Receptor 78 (Olfr78) as a short chain fatty acid (SCFA) receptor which modulates blood pressure. We previously reported that in ex vivo preparations of glomeruli, Olfr78 responds to SCFA's such as propionate to mediate renin release in the juxtaglomerular apparatus (JGA) in the kidney. Similarly, whole‐animal Olfr78 KO mice have lowered plasma renin levels, and are hypotensive.(Pluznick, Protzko et al. 2013). While it is known that Olfr78 is a G‐protein coupled receptor, the precise signaling pathway linking SCFA binding and renin release is not known. In this study, we aimed to establish a cell model which could recapitulate Olfr78‐mediated renin release. To do this, we utilized As4.1 cells, a juxtaglomerular cell line. We found that this cell line does not natively express Olfr78 by RT‐PCR; therefore we studied As4.1 cells either with or without stable expression of a Lucy‐ FLagRho‐Olfr78 construct. (Lucy and Rho tags have been previously shown by our group and others to promote the stable ectopic expression of olfactory receptors). As4.1 release renin upon treatment with relevant stimuli, including the beta‐agonist isoproterenol. We first established methods by which we can detect and quantify this renin release including immunofluorescence, live‐cell imaging, and media fluorescence. As4.1 cells treated with isoproterenol (100μM, 20min) showed a decrease in intracellular renin staining via immunofluorescence as quantified by ImageJ (−35%, n=3, p<0.05). Live cell imaging with quinacrine, an acidotrophic dye that naturally accumulates in renin‐containing granules, also shows a decrease in the fluorescence of these granules after treatment with 100μM isoproterenol (20 min, 30% decrease, n=3, p<0.05). Additionally, we were able to detect renin release by measuring quinacrine fluorescence in the media after treatment of cells with isoproterenol. Media from As4.1 cells had a fluorescence increase from a relative baseline of 1.00 +/− 0.02 in untreated samples to 1.18 +/− 0.02 after isoproterenol treatment (100uM, 10 min, n=8 p<0.05). After establishing these methods, we tested whether we could also quantify renin release in As4.1 cells that stably express Olfr78 following propionate treatment. Media from cells expressing Olfr78 had a fluorescence increase from a relative baseline of 1.00 +/− 0.02 in untreated samples to 1.21+/−0.023 after treatment with propionate (20mM, 10 min, n=8, p<0.05) as compared to wild‐type AS4.1 cells 1.02+/−0.02 (n=8, p>0.05). We hypothesize that Olfr78‐mediated renin release requires increases in cAMP levels, and that this release is inhibited by the elevation of cytosolic calcium levels. Efforts are underway to test this hypothesis and determine the essential downstream components of Olfr78‐ mediated renin release in this cell model, which will provide insights into renin release and blood pressure regulation in vivo.Support or Funding InformationSupport: NIDDKThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intracellular Renin Inhibits Mitochondrial Permeability Transition Pore via Activated Mitochondrial Extracellular Signal-Regulated Kinase (ERK) 1/2 during Ischemia in Diabetic Hearts.

Although beneficial effects of non-secreting intracellular renin (ns-renin) against ischemia have been reported, the precise mechanism remains unclear. In this study, we investigated the roles of ns-renin and mitochondrial extracellular signal-related kinase (ERK) 1/2 on mitochondrial permeability transition pore (mPTP) opening during ischemia in diabetes mellitus (DM) hearts. When isolated hearts from Wistar rats (non-DM hearts) and Goto-Kakizaki rats (DM hearts) were subjected to ischemia for 70 min by left anterior descending coronary artery ligation, DM hearts exhibited higher left ventricular (LV) developed pressure and lower LV end-diastolic pressure than non-DM hearts, suggesting ischemic resistance. In addition, DM hearts showed increased intracellular renin (int-renin, including secreting and non-secreting renin) in the ischemic area, and a direct renin inhibitor (DRI; aliskiren) attenuated ischemic resistance in DM hearts. ERK1/2 was significantly phosphorylated after ischemia in both whole cell and mitochondrial fractions in DM hearts. In isolated mitochondria from DM hearts, rat recombinant renin (r-renin) significantly phosphorylated mitochondrial ERK1/2, and hyperpolarized mitochondrial membrane potential (ΔΨm) in a U0126 (an inhibitor of mitogen-activated protein kinases/ERK kinases)-sensitive manner. R-renin also attenuated atractyloside (Atr, an mPTP opener)-induced ΔΨm depolarization and Atr-induced mitochondrial swelling in an U0126-sensitive manner in isolated mitochondria from DM hearts. Furthermore, U0126 attenuated ischemic resistance in DM hearts, whereas it did not alter the hemodynamics in non-DM hearts. Our results suggest that the increased int-renin during ischemia may inhibit mPTP opening through activation of mitochondrial ERK1/2, which may be involved in ischemic resistance in DM hearts.

O16-1 - Intracellular Renin Inhibits Mitochondrial Permeability Transition Pore via an Activated Mitochondrial ERK1/2 during Ischemia in Diabetic Hearts

O16-1 - Intracellular Renin Inhibits Mitochondrial Permeability Transition Pore via an Activated Mitochondrial ERK1/2 during Ischemia in Diabetic Hearts

Intracellular renin increases the inward calcium current in smooth muscle cells of mesenteric artery of SHR. Implications for hypertension and vascular remodeling

The influence of intracellular renin on the inward calcium current in isolated smooth muscle cells from SHR mesenteric arteries was investigated. Measurements of calcium current were performed using the whole cell configuration of pCLAMP. The results indicated that: 1) renin (100nM) dialyzed into smooth muscle cells, increased the inward calcium current; 2) verapamil (10–9M) administered to the bath inhibited the effect of renin on the inward calcium current; 3) concurrently with the increase of calcium current a depolarization of 6.8+/−2.1mV (n=16)(P<0.05) was found in cells dialyzed with renin; 4) intracellular dialysis of renin (100nM) into smooth muscle cells isolated from mesenteric arteries of normal Wystar Kyoto rats showed no significant change on calcium current; 5) aliskiren (10–9M) dialyzed into the cell together with renin (100nM) abolished the effect of the enzyme on the calcium current in SHR; 6) Ang II (100nM) dialyzed into the smooth muscle cell from mesenteric artery of SHR in absence of renin, decreased the calcium current-an effect greatly reduced by valsartan (10–9M) added to the cytosol; 7) administration of renin (100nM) plus angiotensinogen (100nM) into the cytosol of muscles cells from SHR rats reduced the inward calcium current; 8) extracellular administration of Ang II (100nM) increased the inward calcium current in mesenteric arteries of SHR. Conclusions: intracellular renin in vascular resistance vessels from SHR due to internalization or expression, contributes to the regulation of vascular tone and control of peripheral resistance-an effect independently of Ang II. Implications for hypertension and vascular remodeling are discussed.

Abstract P061: Selective Deletion of Intracellular Renin in the Brain Causes Hypertension and Elevated Sympathetic Nervous System Activity

Renin expression is regulated by two distinct promoter-1st exon combinations that target renin either for secretion (exon 1a for secreted renin) or cytoplasmic retention (exon 1b for intracellular renin, icREN). We developed icREN knockout (KO) mice by selectively deleting exon 1b. icREN KO mice are essentially brain-specific knockouts of icREN because icREN is predominantly expressed in the brain. Notably, systolic blood pressure measured by telemetry was increased in icREN KO mice (130±2 mmHg, n=8 vs 122±2 mmHg in controls, n=7, P&lt;0.01). The low- to high-frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability, a parameter of sympathetic nerve activity (SNA), was increased in icREN KO mice (KO: 1.24±0.21, n=7 vs control: 0.70±0.11, n=7, P&lt;0.05). Body weight (BW) was normal in icREN KO mice compared to controls, but the BW gain and fat accumulation induced by high fat diet (HFD) were attenuated in male icREN KO mice (BW at 16 wks of HFD- KO: 36.8±1.2 g, n=8 vs control: 41.9±1.4 g, n=9; relative fat mass at 14 wks of HFD- KO: 27.7±1.7%, n=8 vs control: 34.4±2.3%, n=9, both P&lt;0.05). The resting metabolic rate measured by respirometry was increased in icREN KO mice (0.156±0.005 kcal/h, n=46, P&lt;0.05) vs controls (0.145±0.003 kcal/h, n=53), whereas food consumption and absorbed calories were not different. We previously reported that the brain renin-angiotensin system facilitates renal SNA (RSNA) response to acute intracerebroventricular (ICV) injection of leptin. Interestingly, the RSNA response to ICV leptin was greater in icREN KO mice (KO: 214±40 % baseline, n=5 vs control: 114±18 % baseline, n=10, P&lt;0.01). AT1a receptor mRNA was upregulated in the paraventricular nucleus of icREN KO mice (P&lt;0.05). Chronic ICV injection of losartan not only abolished the elevated blood pressure in icREN KO mice, but reduced it to below baseline in controls (systolic blood pressure, 111±3 mmHg in KO, n=5; 124±4 mmHg in controls, n=6). These data suggest that icREN deletion increases the activity of brain renin-angiotensin system and elevates blood pressure and metabolic rate through sympathetic activation. We conclude that this novel icREN isoform contributes to cardiovascular and metabolic control possibly as part of an inhibitory neural circuit.

Chemical Communication between Heart Cells is Disrupted by Intracellular Renin and Angiotensin II: Implications for Heart Development and Disease.

HighlightsIntracellular renin and angiotensin disrupts chemical communication in heart.Epigenetic modification of renin angiotensin aldosterone system (RAAS) and heart disease.Intracrine renin angiotensin and metabolic cooperation.Gap junction, intracellular renin and angiotensin, cellular patterns, and heart development. The finding that intracellular renin and angiotensin II (Ang II) disrupts chemical communication and impairs metabolic cooperation between cardiomyocytes induced by aldosterone, hyperglycemia, and pathological conditions like myocardial ischemia is discussed. The hypothesis is presented that epigenetic changes of the renin angiotensin aldosterone system (RAAS) are responsible for cardiovascular abnormalities, including the expression of RAAS components inside cardiac myocytes (intracrine RAAS) with serious consequences including inhibition of electrical and chemical communication in the heart, resulting in metabolic disarrangement and cardiac arrhythmias. Moreover, the inhibition of gap junctional communication induced by intracellular Ang II or renin can contribute to the selection of cellular patterns during heart development.

Intracellular Renin in the Brain Contributes to Cardiovascular and Metabolic Control

Renin gene expression is regulated by two distinct promoter‐first exon combinations that target renin either for secretion (exon 1a initiates secreted renin) or cytoplasmic retention (exon 1b initiates intracellular renin, icREN). Interestingly, icREN is expressed predominantly in the brain. We selectively deleted exon 1b and developed icREN KO mice to examine the role of icREN in cardiovascular and metabolic control. Systolic blood pressure in the light phase as measured by telemetry was increased in KO mice (126±2 mmHg, n=8 vs 116±2 mmHg, n=7, P&lt;0.01). The low‐ to high‐frequency ratio (LF/HF) derived from power spectral analysis of heart rate variability was increased in KO mice (1.24±0.21, n=7 vs 0.70±0.11, n=7, P&lt;0.05). Body weight was normal in KO mice fed normal chow. However, high fat diet‐induced obesity was attenuated in male KO mice (body weight, 32.4±1.3 g, n=5 vs 36.6±1.4 g, n=9; relative fat mass, 26.1±2.3 %, n=5 vs 34.4±2.3 %, n=9, both P&lt;0.05). The resting metabolic rate measured by respirometry was increased in KO mice (0.156±0.005 kcal/h, n=46, P&lt;0.05) vs controls (0.145±0.003 kcal/h, n=53). We previously reported that the brain renin‐angiotensin system facilitates renal sympathetic nerve activity (RSNA) response to acute intracerebroventricular injection of leptin. Interestingly, the RSNA response to leptin was greater in KO mice compared to controls (214±40 % baseline, n=5 vs 114±18 % baseline, n=10, P&lt;0.01). Further, in KO mice, AT1a receptor mRNA was upregulated in the paraventricular nucleus (P&lt;0.05). These data suggest that this novel icREN isoform contributes to cardiovascular and metabolic control possibly through the modulation of AT1a receptor expression in the brain.

Intracellular Renin Disrupts Chemical Communication between Heart Cells. Pathophysiological Implications.

Highlights Intracellular renin disrupts chemical communication in the heartAngiotensinogen enhances the effect of reninIntracellular enalaprilat reduces significantly the effect of reninIntracellular renin increases the inward calcium currentHarmful versus beneficial effect during myocardial infarctionThe influence of intracellular renin on the process of chemical communication between cardiac cells was investigated in cell pairs isolated from the left ventricle of adult Wistar Kyoto rats. The enzyme together with Lucifer yellow CH was dialyzed into one cell of the pair using the whole cell clamp technique. The diffusion of the dye in the dialyzed and in non-dialyzed cell was followed by measuring the intensity of fluorescence in both cells as a function of time. The results indicated that; (1) under normal conditions, Lucifer Yellow flows from cell to cell through gap junctions; (2) the intracellular dialysis of renin (100 nM) disrupts chemical communication – an effect enhanced by simultaneous administration of angiotensinogen (100 nM); (3) enalaprilat (10−9 M) administered to the cytosol together with renin reduced drastically the uncoupling action of the enzyme; (4) aliskiren (10−8 M) inhibited the effect of renin on chemical communication; (5) the possible role of intracellular renin independently of angiotensin II (Ang II) was evaluated including the increase of the inward calcium current elicited by the enzyme and the possible role of oxidative stress on the disruption of cell communication; (6) the possible harmful versus the beneficial effect of intracellular renin during myocardial infarction was discussed; (7) the present results indicate that intracellular renin due to internalization or in situ synthesis causes a severe impairment of chemical communication in the heart resulting in derangement of metabolic cooperation with serious consequences for heart function.

Intracellular Renin Protects Cardiomyocytes from Ischemic Injury in Diabetic Heart

Background: Local Renin-Angiotensin-Aldosterone system (RAS) is important in cardiac pathophysiology. We investigated the expression and distribution of intracellular renin after ischemia, and the effects of renin on mitochondrial function in diabetic heart. Methods: In Goto-Kakizaki (DM) and Wistar (non-DM) rats, Langendorff-perfused hearts were subjected to ischemia by coronary artery ligation for 90 min. Infarct Size (IS) and expression of RAS components were examined. Mitochondrial membrane potential (ΔΨm), uncoupling protein-2 (UCP2), NAD/NADH ratio, and ATP were measured in renin-treated myocytes or isolated mitochondria. Results: After ischemia, LV function (LVDP; 76 ± 4 vs. 52 ± 4 mmHg, LVEDP; 18 ± 2 vs. 29 ± 4 mmHg, p<0.05, DM; n=9, non-DM; n=9, respectively) was prevented and IS (44.2 ± 2.1 vs. 53.7 ± 2.9 %, p<0.05) was significantly small in DM hearts. These cardioprotective effects were abolished when DM hearts were treated with direct renin inhibitor (LVDP; 77 ± 3 vs. 55 ± 4 mmHg, LVEDP; 16 ± 1 vs. 27 ± 3 mmHg, IS; 40.8 ± 2.9 vs. 52.1 ± 3.4 %, p<0.05, DM; n=5, DM plus DRI; n=5, respectively). Renin expression in the ischemic area was increased in DM hearts. Electron microscopy showed predominant renin localization within mitochondria. In permeabilized myocytes or isolated mitochondria, renin hyperpolarized ΔΨm, increased NAD/NADH ratio and preserved ATP content. Ischemiainduced UCP2 expression was reduced in DM. Conclusions: Intracellular renin, which mainly localizes within mitochondria, increased during ischemia and protected cardiomyocytes in diabetic hearts. This protective effect of renin is at least partially because of the reduction of UCP2 and the acceleration of electron transport chain, which resulted in the prevention of mitochondrial depolarization and ATP production.

Abstract 080: Selective Knockout of the Intracellular Isoform of Renin in the Brain Contributes to Metabolic and Cardiovascular Control

Renin gene expression is regulated by two distinct promoter-first exon combinations that target renin for either secretion (exon 1a initiating secreted renin, sREN) or for cytoplasmic retention (exon 1b initiating intracellular renin, icREN). The icREN isoform is expressed predominantly in the brain and its expression is downregulated whereas sREN expression is upregulated by deoxycorticosterone (DOCA)-salt suggesting each isoform may be differentially regulated. We generated mice that lack icREN, but preserve sREN, by flanking exon-1b and its surrounding sequences (including the promoter) with loxP sites and breeding successively with mice expressing flipase and cre-recombinase. Real time quantitative RT-PCR analysis revealed a loss of icREN mRNA in the brain, but a preservation of sREN mRNA in the kidney. Total body weight was normal in male icREN-KO mice (26.3±0.7 g, n=8, 12 wk) compared to controls (25.3±0.7 g, n=12, 12 wk), but total (2.14±0.09 g vs 2.46±0.11 g) and relative (9.0±0.5 % vs 10.7±0.6%) fat mass as measured by NMR were reduced in 8-week icREN-KO mice (n=14-16, P&lt;0.05). Whereas food intake was normal, there was an increase in resting metabolic rate as measured by respirometry in icREN-KO (0.1484±0.0036 kcal/hr, n=41, P=0.02 by ANCOVA) vs controls (0.1389±0.0038 kcal/hr, n=38). There was also a trend toward decreased digestive efficiency (81.1±1.0%, n=6, P=0.06) vs controls (84.9±1.2%, n=3) at 22 wk of age. Systolic blood pressure was decreased in icREN-KO mice (113±1 mmHg, n=12, 17.0±1.2 wk vs 120±3 mmHg, n=5, 15.9±1.9 wk, P&lt;0.05). We previously reported a functional link between the synthesis and action of angiotensin-II and the action of leptin. We tested if this was impaired in icREN-KO mice by measuring the renal sympathetic nerve activity (RSNA) response to acute intracerebroventricular (ICV) administration of leptin. The decrease in arterial pressure was confirmed in this cohort of icREN-KO mice. Surprisingly, the RSNA response to leptin was greater in icREN-KO mice compared to controls (257.9±16.0%, n=3 vs 157.0±13.9%, n=4, 4 hr after leptin, P=0.01). Captopril attenuated the sympathetic response to ICV leptin. Together these data suggest that this novel icREN isoform contributes to metabolic and cardiovascular control.

Renin inhibitor VTP-27999 differs from aliskiren

VTP-27999 is a renin inhibitor with an IC50 that is comparable to that of aliskiren, but with a higher bioavailability. Unexpectedly, VTP-27999, unlike aliskiren, did not unfold renin's precursor, prorenin, and increased the affinity of the antibodies applied in renin immunoassays. Here we verified to what degree these differences affect intracellular renin inhibitor accumulation in renin-synthesizing human mast cells (HMC-1), and (pro)renin's signaling via the (pro)renin receptor ((P)RR) in rat vascular smooth muscle cells. We also addressed the consequences of (P)RR knockdown by small-interfering (si) RNA on (pro)renin release. Finally, making use of FRET(Bodipy-FL)-labeled aliskiren, we studied, by subcellular fractionation, the cellular distribution pattern of this renin inhibitor. VTP-27999 accumulated at higher levels in HMC-1 cells than aliskiren, allowing this inhibitor to block intracellular renin at approximately five-fold lower medium levels. Labeled aliskiren accumulated in mitochondria and lysosomes, and its distribution pattern was different from that of renin. Moreover, the intracellular accumulation of both inhibitors in nonrenin-synthesizing HEK293 cells was not different from that in HMC-1 cells, suggesting that it is renin synthesis-independent. VTP-27999, but not aliskiren, blocked renin's capacity to stimulate extracellular signal-regulated kinase 1/2 phosphorylation in vascular smooth muscle cells, whereas neither inhibitor interfered with prorenin-induced signaling. (P)RR knockdown greatly increased renin (and to a lesser degree, prorenin) release, without affecting the capacity of forskolin or cAMP to stimulate renin release. VTP-27999 differs from aliskiren regarding its level of intracellular accumulation and its capacity to interfere with renin signaling via the (P)RR, and the (P)RR determines prorenin-renin conversion and constitutive (but not regulated) (pro)renin release.

Potential role of AT2R on the cardiac mitochondrial metabolism in the hyperthyroidism condition (1159.8)

Thyroid hormone (TH) is known to influence the mitochondrial metabolism. Recently, it has been proposed that the intracellular renin angiotensin system (iRAS) seems to be activated by high levels of (TH). However, the contribution of AT2R in the regulation of cardiac mitochondrial metabolism or the possible effect of TH on this event was not elucidated yet. We investigated the potential role of AT2R on the cardiac mitochondrial metabolism and the possible influence of the hyperthyroidism on this process. Four groups of mice were used: wild‐type (WT), WT treated with TH (0.7 µg/Kg BW/day, i.p.) for 14 days, knockout (KO AT2R‐/y) mice and KO AT2R‐/y mice treated with TH. Cardiac mitochondria were isolated and the oxygen consumption was assessed by oxymetry. The H2O2 production was also analyzed. The determination of maximal oxygen uptake was determined by indirect calorimetry open circuit. Data were analyzed by two‐way ANOVA. The KO mice presented a reduction in all parameters analyzed: states 2, 3, 4 and respiratory control ratio in relation to control. The TH‐treated WT did not present changes on H2O2 production. However, H2O2 production was higher on KO mice treated with TH. Indeed, the calorimetry showed a decrease (17%) on basal metabolic rate of on KO mice. Taken together, our data show that AT2R may be involved in the control of cardiac mitochondrial metabolism and influence the effect of hyperthyroidism in this parameter.Grant Funding Source: Supported by CNPq/CAPES and FAPESP

Abstract 16: Physiological Characterization of a Selective Knockout of the Intracellular Isoform of Renin in the Brain of Mice

Renin gene expression is regulated by two distinct promoter-first exon combinations that target renin for either secretion (exon 1a initiating secreted renin, sREN) or for cytoplasmic retention (exon 1b initiating intracellular renin, icREN). The icREN isoform is expressed predominantly in the brain and its expression is downregulated whereas sREN expression is upregulated by deoxycorticosterone (DOCA)-salt suggesting each isoform may be differentially regulated. We generated mice that conditionally lack icREN, but preserve sREN, by targeting a novel renin allele in ES cells by flanking exon-1b and its surrounding sequences (including the promoter) with loxP sites. Mice carrying this allele were first bred with flippase mice to remove the neo cassette and then with cre-recombinase mice to generate the null allele lacking icREN. The structure of the final allele was confirmed by Southern blot. There was no apparent lethality in the homozygous mice when heterozygous mice were intercrossed. Real time quantitative RT-PCR analysis revealed a loss of icREN mRNA in the brain, but a preservation of sREN mRNA in the kidney. Male icREN-KO mice were similar in size (n=4, 14.7±0.9 wk, 27.94±1.08 g) compared to littermate controls (n=7, 14.2±0.8 wk, 27.96±0.82 g, P=0.988), but by NMR exhibited reduced total and relative total fat mass (1.83±0.15 vs 3.00±0.12 g, or 6.6±0.7 vs 10.9±0.5 %, both P&lt;0.001). Interestingly, interscapular brown adipose (289±77, n=5 vs 68±61 mg, n=9 P=0.04) and heart (162±12 vs 129±9 mg, P=0.04) masses were both increased in icREN-KO mice, possibly suggesting increased resting metabolic rate (RMR) and blood pressure. Liver and kidney masses were normal. Food intake was normal (3.25±0.28, n=4 vs 3.14±0.14, n=8 P=0.7), but preliminary tests uncovered trends toward increased RMR (7.67±0.75, n=4 vs 6.93±0.59 g/day, n=4 kcal/kg lean/hr, P=0.47) and glucose tolerance (2g/kg, 25768±3994, n=3 vs 33734±2854 mg/dL*min, n=8, P=0.157). Studies are ongoing to assess arterial pressure. Together these data suggest that this novel icREN isoform contributes to metabolic and possibly to cardiovascular control. Future studies using the conditional allele (icREN-flox) will provide an opportunity to dissect the neural circuits involved in these responses.

Selective knockout of novel intracellular renin isoform results in reduced adiposity

Renin gene expression is regulated by two distinct promoter / first exon combinations that target renin for either secretion (exon 1a) or for cytoplasmic retention (exon 1b). Renin‐1b is expressed predominantly in the brain and is differentially regulated from renin‐1a in response to stimuli such as deoxycorticosterone (DOCA)‐salt. Mice were generated that lack exon 1b (and thereby intracellular renin, icREN‐KO) but retain exon 1a. Male icREN‐KO mice were similar in size (n=4, 14.7±0.9 wk, 27.94±1.08 g) compared to littermate controls (n=7, 14.2±0.8 wk, 27.96±0.82 g, P=0.988), but by NMR exhibited reduced total and relative total fat mass (1.83±0.15 vs 3.00±0.12 g, or 6.6±0.7 vs 10.9±0.5 %, both P&lt;0.001). Interestingly, interscapular brown adipose (289±77, n=5 vs 68±61 mg, n=9 P=0.04) and heart (162±12 vs 129±9 mg, P=0.04) masses were both increased in icREN‐KO mice, possibly suggesting increased resting metabolic rate (RMR) and blood pressure. Liver and kidney masses were normal. Food intake was normal (3.25±0.28, n=4 vs 3.14±0.14, n=8 P=0.7), but preliminary tests uncovered trends toward increased RMR (7.67±0.75, n=4 vs 6.93±0.59 g/day, n=4 kcal/kg lean/hr, P=0.47) and glucose tolerance (2g/kg, 25768±3994, n=3 vs 33734±2854 mg/dL*min, n=8, P=0.157). Together these data suggest that the novel icREN isoform contributes to metabolic control.

Angiotensin II Stimulates Renin Synthesis and Secretion in Mouse Collecting Duct M‐1 cells via a PKC alpha‐mediated cAMP Stimulation Mechanism

To test the hypothesis that Ang II stimulates renin synthesis and secretion in collecting duct (CD) cells via activation of PKC and subsequent stimulation of cAMP/PKA/CREB pathway, we treated mouse cortical CD (M‐1) cells with Ang II (10−7M). Ang II treatment for 6‐h increased intracellular renin gene expression and renin content in culture media (Ang II: 31±4 vs. 19±4 ng Ang I/hr/ml; p&lt;0.05). Likewise, this treatment increased intracellular cAMP levels by 1 min (148±32 pmol/mg protein) which reached a peak at 30 min (290±27 vs. 44±0 pmol/mg protein; p&lt;0.05). The adenlyate cyclase activator, forskolin and the analogue of cAMP, dibutyryl‐cAMP both significantly increased renin gene expression. These responses were followed by increases in pCREB levels and prevented by PKA inhibition with H89 or PKI. PKC inhibition either with calphostin C or a PKCα dominant negative prevented the augmentation of intracellular levels of cAMP, pCREB and renin mRNA in Ang II‐treated M‐1 cells. Ang II stimulates renin synthesis and secretion in M‐1 cells via PKCα‐mediated cAMP accumulation and PKA/CREB activation. By this mechanism distal tubular renin may contribute to local Ang II generation, thus to activation of sodium reabsorption.