Transitioning from the Bench to the Classroom, an Education-Intensive Career.

s of Invited and Contributed Presentations 1.0 Novel Aspects of the Endothelin System....................................................................13 2.0 The Immune System and Endothelin......................................................................13 3.0 Submitted Abstracts..........................................................................................13 4.0 ET, Sex and Pregnancy.......................................................................................34 5.0 Role of ET in the Vasculature...............................................................................35 7.0 Endothelin and End-Organ Injury .........................................................................35 8.0 Endothelin, Angiotensin and Vascular Function........................................................35 9.0 Endothelin and Fluid Electrolyte Balance..................................................................35 12.0 Novel Integration.............................................................................................35 13.0 Endothelin Therapeutics—Where are we?........................................................................36 Author Index................................................................................................38 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 13 1.0 NOVEL ASPECTS OF THE ENDOTHELIN SYSTEM 1.1 NEW INSIGHTS IN ET RECEPTOR PHARMACOLOGY Janet Maguire, Rhoda Kuc, and Anthony Davenport EMIT, Univ. of Cambridge, Level 6 ACCI, Box 110 Addenbrooke's Hosp., Cambridge, CB2 0QQ, UK. Understanding endothelin receptor pharmacology is essential to unravelling the role of these important peptides in health and disease. Recently, comprehension of how Gprotein coupled receptors (GPCRs) interact with their ligands to transmit extracellular signals into cellular responses has expanded, with some of the basic tenets of pharmacology requiring re-evaluation. This should prompt researchers to look at published data with a new perspective and to rethink the design of future experiments. The two endothelin receptors, ETA and ETB, are defined by their rank order of potency for the three endogenous peptides, ET-1, ET-2 and ET-3. Selective agonists are available for the ETB receptor but, interestingly, not the ETA receptor. Receptor selective and non-selective antagonists have been developed as important research tools for defining receptor function and as clinically significant drugs for pulmonary arterial hypertension. However, some of the pharmacology of the two receptors has been difficult to reconcile, for example differences in ligand affinity for cloned and native receptors and ligand dependence of antagonist affinities. The aim of this talk is to revisit what we know about the pharmacology of endothelin receptors and to reevaluate these data in the light of recent structural studies and the discovery of GPCR biased signalling. 1. Rosenbaum DM, Rasmussen SG, Kobilka BK. 2009. The structure and function of G-protein-coupled receptors. Nature 459:356–363. 2. Wisler JW, Xiao K, Thomsen AR, Lefkowitz RJ. 2014. Recent developments in biased agonism. Curr Opin Cell Biol 27:18–24. 2.0 THE IMMUNE SYSTEM AND ENDOTHELIN 2.1 INFLAMMATION, IMMUNITY AND HYPERTENSION David Harrison Internal Med., Vanderbilt Univ., 2220 Pierce Ave, Nashville, TN, 37232-6602. Hypertension remains an enormous health care burden that affects 30% of Western populations. Despite its prevalence the cause of most cases of hypertension remain unknown. Our laboratory has defined a novel mechanism for hypertension involving adaptive immunity. We found that mice lacking lymphocytes (RAG-1 mice) develop blunted hypertensive responses to a variety of stimuli including chronic angiotensin II infusion, DOCA-salt challenge and norepinephrine infusion. Adoptive transfer of T cells, but not B cells, restores the hypertensive responses to these stimuli. Hypertension is associated with the infiltration of T cells into the kidney and vasculature, where they release cytokines, including IFN-γ, IL-17A, and TNFα, which promote sodium retention, vasoconstriction and oxidative injury. Recently, we have found that angiotensin II has striking effects on dendritic cells (DCs), promoting their propensity to activate T cells. Our data indicate that angiotensin II infusion increases DC superoxide production by 5-fold and causes a striking accumulation isoketals, oxidized products of arachidonic acid in these cells. These form covalent bonds to lysines of proteins and these modified proteins become immunogenic. Several isoketal scavengers, including 2-hydroxybenzylamine (2-HOBA) prevent DC activation, the ability of DCs to stimulate T cell proliferation and prevent hypertension. A major impetus for immune cell activation seems to be increased sympathetic outflow, stimulated by the central actions of angiotensin II. By lesioning the AV3V region of the forebrain of mice or inactivating the NADPH oxidase in the subfornical organ using Cre Lox technology, we have prevented the central actions of angiotensin II and found that this inhibits both T cell activation and hypertension. Renal denervation likewise prevents activation of DCs in the kidney and the accumulation of activated DCs in the spleen. Thus, the kidney seems to be a major site of DC activation in hypertension. In summary, we have identified a new mechanism underlying hypertension and a potential new therapy for this common and yet difficult to manage disease. 3.0 POSTERS 3.1 RENAL VASCULAR REGENERATION BY ANGIOTEN-SIN II ANTAGONISM IS DUE TO ABROGATION OF ET-1/ETAR SIGNALING Ariela Benigni, Andrea Remuzzi, Fabio Sangalli, Daniela Mac-coni, Susanna Tomasoni, Irene Cattaneo, Paola Rizzo, Barbara Bonandrini, Elena Bresciani, Lorena Longaretti, Elena Gagliar-dini, Sara Conti, and Giuseppe Remuzzi Centro Anna Maria Astori, IRCCS-Inst. di Ricerche Farmacologiche Mario Negri, Via Stezzano, 87, Bergamo, Italy, Dept. of Industrial Engineering, Bergamo Univ., Viale Marconi, 5, Dalmine, Bergamo, Italy, Unit of Nephrology and Dialysis, Azienda Ospedaliera Papa Giovanni XXIII, Piazza OMS, 1, Bergamo, Italy. Endothelial dysfunction and vascular rarefaction play an important role in the progression of renal fibrosis. Treatment of Munich Wistar Fronter (MWF) rats with advanced nephropathy with an angiotensin converting enzyme inhibitor showed regression of established renal lesions and substantial glomerular tuft repair. Here we investigated whether this effect was associated with protection of the kidney vascularture. The whole kidney vasculature was analyzed by micro-computed tomography (microCT) in untreated and lisinoprilor losartan-treated MWF rats and in Wistar rats as controls. Drugs were given at 50 week-old animals with established renal damage for 10 weeks. The 3D reconstruction of the vascular network showed a progressive rarefaction affecting intermediate and small size vessels in kidneys from aged MWF rats as compared to controls. These changes were associated with endothelial mesenchymal transition (EndMT) and apoptosis concomitant with the overexpression of pro-fibrotic genes including endothelin-1 (ET-1). Within the glomerulus, ET-1 protein was highly expressed by both endothelial cells (EC) and podocytes as documented by co-staining of RECA-1 and a-actinin-4. Renal ETAR expression in the vascular endothelium of MWF rats was also increased in a time-dependent manner. Renin angiotensin system (RAS) inhibition halted vascular rarefaction and even increased the volume density of kidney vessels as compared to pre-treatment suggesting a regenerative process. The treatment normalized ET-1/ ETAR renal endothelial expression and significantly reduced EndMT and apoptosis while increased EC proliferation. Our data suggest that ET-1/ ETAR deregulation contribute to renal EC damage and vascular rarefaction and that restoration of total kidney vasculature by RAS inhibition relates in part to abrogation of ET-1/ ETAR signaling pathway. 3.2 REGULATION OF COLLECTING DUCT ENDOTHELIN-1 PRODUCTION BY FLOW AND OSMOLALITY Yang Gao, Meghana Pandit, and Donald Kohan Div. of Nephrology, Univ. of Utah, 1900 E. 30 N., Salt Lake City, UT, 84132. Background: Endothelin-1 (ET-1) produced by the renal collecting duct (CD) is an important regulator of blood pressure and urinary sodium and water excretion. CD ET-1 production is increased by high salt intake; since ET-1 acts as an autocrine inhibitor of CD sodium and water reabsorption, this process facilitates normalization of body fluid volume. The mechanisms coupling salt intake to CD ET-1 synthesis are incompletely understood. Herein, we have investigated the role of tubule fluid flow and tubule fluid solute delivery in stimulating CD ET-1 production since both of these factors are augmented by a high salt diet. Methods: A mouse inner medullary collecting duct cell line (IMCD3) was exposed to stationary conditions or laminar flow (using Hanks Balanced Salt Solution) at a shear stress of 2 dyne/cm for 2 hr at 37C (conditions determined to maximize the ET-1 flow response). The ratio of IMCD3 ET-1 to GAPDH mRNA levels was determined; since ET-1 protein is below detection levels due to the small number of cells and since ET-1 mRNA almost always parallels ET-1 protein levels, ET-1 mRNA content was taken as an index of ET-1 ET-1 protein levels. For all studies, N≥10 per data point. Results: ET-1 mRNA increased by 219 ± 21% in response to flow (compared to cells not exposed to flow). When perfusate osmolality was increased from 300 to 450 mOsm/L with NaCl, urea or mannitol, the ET-1 flow response increased to 450-500% over that seen in cells not exposed to flow (but containing 450 mOsm/L). This heightened flow response to osmolality was not altered by inhibition of the epithelial sodium channel (using 0.2 μM benzamil). While the ET-1 flow response under 300 mOsm/L conditions was blocked by chelation of intracellular calcium (50 μM BAPTA-AM), calcineurin inhibition (3 μg/ml cyclosporine A), purinergic receptor blockade (30 μM PPADS), or genetic deletion of polycystin-2, the augmented flow response in the presence of increasing solutes to 450 mOsm/L was not affected by these maneuvers. In contrast, inhibition of NFAT5 with 10 μM rottlerin abolished the ET-1 flow response under 300 or 450 mOsm/L conditions. Since rottlerin can have off-target effects, more specific evaluation of NFAT5 was performed. NFAT5 siRNA (68% knockdown of NFAT5 mRNA) completely blocked the heighted ET-1 flow response seen with 450 mOsm/L perfusate. Conclusions: Tubule fluid flow increases IMCD ET-1 production via a calcium, calcineurin, purinergic receptor and polycystin-dependent mechanism. Increased perfusate osmolality increases the IMCD ET-1 flow response via an NFAT5-dependent pathway. The flow and osmolality pathways work in concert to augment CD ET-1 production, providing evidence that both tubule fluid flow and solute delivery are involved in augmenting CD ET-1 production in response to salt loading. Funding source: NIH P01 HL095499. 3.3 THE ROLE OF ENDOTHELIN SYSTEM IN RENAL STRUCTURE AND FUNCTION DURING THE POST-NATAL DEVELOPMENT OF THE RAT KIDNEY M. F. Albertoni Borghese, M. C. Ortiz, S. Balonga, A. Lavagna, A. Filipuzzi, M. Barchuk, A. Schneider, R. Moreira Szokalo, and M. Majowicz 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 14 Cellular & Molecular Biology, Dpt. Biological Sci., Sch. of Pharmacy & Biochemistry, Univ. of Buenos Aires, Junin 956 1st Fl., Buenos Aires, 1113, Argentina. Renal development in rodents, unlike in humans, continues during early postnatal period. We aimed to evaluate whether the pharmacological inhibition of ET system during this period affects renal development, both at structural and functional level in male and female rats. Newborn rats were treated orally from postnatal day 1 to 20 with vehicle or bosentan (Actelion, 20 mg/kg/day), a dual endothelin receptor antagonist (ERA). The animals were divided in 4 groups: control males (Cm), control females (Cf), ERA males (ERAm) and ERA females (ERAf). At day 21, one kidney was used to assess the glomerular number by a maceration method, and the other was used to perform morphometric analysis with Image Pro Plus software. Results are mean ± SEM (n ≥ 6). Two-way ANOVA was used for the statistical analysis. The body weight of ERAm and ERAf decreased when compared with Cm and Cf respectively. However, neither femur length nor kidney weight/100g bw showed differences between groups. The number of total glomeruli (maceration method) decreased in ERAm vs Cm (Cm: 101499 ± 3526; ERAm: 84734 ±2709*; Cf: 89225±7032; ERAf: 88762 ±3359). The morphometric evaluation showed that the number of glomeruli/mm2 decreased in the juxtamedullary (JM) area in ERAm and ERAf vs Cm and Cf respectively (Cm: 12.9±0.8; ERAm: 10.2±0.8**; Cf: 13.4±0.9; ERAf: 11.2±0.9##). The JM renal filtration surface area (μm) decreased in ERA groups (Cm: 55406±3496; ERAm: 44297±3720*; Cf: 61697±5208*; ERAf: 52496±4108#&). There was a decrease in the ratio Capilar Glomerular Area/Total Glomerular Area (%) of the JM nephrons in ERAf, whereas in ERAm there was a tendency to decrease this parameter (Cm: 80.5±0.9; ERAm: 78.7±1.0; Cf: 83.5±1.4; ERAf: 75.7±0.8###). There were no changes in the same parameters in the cortical area; although there was a tendency to decrease those parameters in ERAm and ERAf. The creatinine clearance (ml/min/100g), decreased in ERAm and ERAf vs Cm and Cf respectively (Cm= 0,33±0,03; ERAm: 0,26±0,02*; Cf: 0,34±0,04; ERAf: 0,24±0,04#). There was an increase in proteinuria (mg/24h/100g) in ERAm and ERAf vs Cm and Cf respectively (Cm: 1.96 ± 0.30; ERAm: 3.16±0.29*; Cf: 1.92±0.32; ERAf: 2.40±0.39#) and a tendency to increase diuresis in ERA groups. *p<0.05, **p<0.01 vs Cm; #p<0.05, ## p<0.01, ### p<0.005 vs Cf; &p;<0.05 vs ERAm. These results suggest that ET has an important role in rat renal postnatal development. The observed decrease in nephron number and the increase in proteinuria are factors associated with susceptibility to develop hypertension and renal diseases. However, these results do not imply that the same could happen in humans, since human renal development is complete at birth. This work was supported by UBACyT grant 20020130200184BA and 20020090200685. 1. Murawski; 2010. 2. Loria; 2007. 3.4 THE ROLE OF ENDOTHELIN IN THE REGULATION OF BLOOD PRESSURE IN EARLY DIABETES MELLITUS Geoff Culshaw, Matthew Bailey, Patrick Hadoke, and David Webb Queen's Med. Res. Inst., Univ. of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK. Introduction: Diabetes mellitus (DM) is associated with sodium and water retention, loss of diurnal variation in arterial blood pressure (BP), and hypertension. Endothelin1 (ET-1) regulates BP by vasoconstriction via ETA receptors and natriuresis via ETB receptors in the renal collecting duct. We hypothesised that in early DM, BP is increased and diurnal variation in BP is lost through enhanced ETA receptor signalling and reduced ETB receptor signalling. Methods: Heart rate (HR), systolic, diastolic and mean BPs (SBP, DBP, MBP) and diurnal variation in BP were recorded continuously by radiotelemetry in 8 adult male Sprague Dawley rats (322g SEM 4). Recording started 2 weeks after intraperitoneal injection of vehicle or streptozotocin (3045mg/kg) to induce DM (n=4 per group). There were 4 recording periods of 7 days: baseline, salt supplementation, salt + ETA antagonist (oral atrasentan 5mg/kg/day), and salt + ETA (atrasentan) and ETB antagonist (oral A-192621 10mg/kg/day). Results: At baseline, diabetic rats had higher DBP (97±1mmHg) and MBP (110±1mmHg) than controls (93±1mmHg and 108±1mmHg respectively; P<0.001). Otherwise, BP did not differ between the 2 groups during the whole study despite a lower HR in diabetics (326±2 beats/min vs 354±2 beats/min control; P<0.001). BP in diabetics was unaffected by salt supplementation. In controls, salt increased SBP (126±1mmHg to 130±1mmHg; P<0.001) and MBP (107±1mmHg to 112±1mmHg; P<0.001). ETA antagonism reduced SBP, DBP and MBP in both groups (all P<0.001) with no difference in the effect size. Reductions in BP were countered by ETB antagonism (all P<0.001). Diurnal dipping in BP was less in diabetic rats than controls at baseline (DBP 2.3% vs 7.2%, P <0.001; MBP 2.7% vs 5.1%, P=0.011) and during ETA antagonism (DBP 1.4% vs 4.4%, P=0.006; MBP 1.5% vs 3.3%, P=0.06). 24 hour periodicity in DBP and MBP was lost in diabetics but not controls (DBP P<0.001; MBP P<0.044) during ETA antagonism, but was present in both groups during mixed ET receptor antagonism (all P<0.044). Conclusion: BP was increased and diurnal variation in BP was reduced in this model of early DM. Selective ETA antagonism, but not mixed ET receptor antagonism, reduced BP in controls and diabetic rats but did not recapitulate diurnal variation in BP in diabetics. ETA receptors but not ETB receptors may represent a therapeutic target for hypertension in early DM. Funded by Kidney Research UK, The British Heart Foundation and The Roslin Institute. 3.5 TUDCA ATTENUATES HIGH SALT-INDUCED RENAL CORTICAL INJURY IN ETB RECEPTOR DEFICIENT RATS BY DECREASING APOPTOSIS Randee Sedaka, Carmen De Miguel, Janet L. Hobbs, David M. Pollock, and Jennifer S. Pollock Section of Cardio/Renal Physiology & Med., Dept. of Med./Nephrology, Univ. of Alabama at Birmingham, Kaul 830, 720 20th St. S, Birmingham, AL, 35294. ETB receptor deficient (ETB def) rats have a loss of functional ETB receptors with higher circulating ET-1 displaying salt-sensitive hypertension and renal injury. It is unknown if apoptosis via cellular stressors are involved in high salt-induced renal injury. We hypothesized that high salt-induced renal injury in ETB def rats occurs via apoptosis. The chemical chaperone, tauroursodeoxycholic acid (TUDCA), nullifies cellular stress pathways. To test our hypothesis, we utilized ETB def and transgenic (TG) control rats on normal (1% NaCl, NSD) or high salt (8% NaCl, HSD) diet for three weeks (n=5-7/group) given daily TUDCA (400 mg/kg/day; i.p.) or vehicle. TUDCA did not influence blood pressure in either group. Urinary renal injury biomarkers (NGAL, KIM-1, albumin, nephrin) were assessed. HSD significantly increased renal injury in ETB def rats (NSD vs. HSD; KIM-1: 14.2±3.4 vs. 104.1±20.6 pg/day; NGAL: 43.6±17.6 vs. 215.6±27.7 pg/day; albumin: 0.12±0.04 vs. 10.69±3.43 ng/day, p<0.001), while HSD did not elicit any change in TG rats. TUDCA significantly decreased injury markers (KIM-1: 55.7±13.8 pg/day; NGAL: 114.2±18.0 pg/day; albumin: 2.27±1.54 ng/day, p<0.05) in ETB def rats. Renal cortical tissue KIM-1 levels mirrored the urinary KIM-1 excretion in ETB def rats (KIM-1: NSD 10.2±3.0 pg/day vs. HSD 77.5±8.8 pg/day vs. TUDCA 25.4±7.6 pg/day, p<0.001). Renal glomerular injury assessed by nephrin excretion significantly increased in both ETB def and TG rats on HSD, however TUDCA failed to attenuate this increase in either group. Apoptosis was increased in the renal cortex of ETB def rats on HSD (NSD vs. HSD: 4.0±1.0 vs. 18.8±4.2 TUNEL cells/field), while TUDCA decreased the high salt-induced apoptosis (2.1±0.1 TUNEL cells/field). No significant apoptosis was detected in the renal cortex in TG rats. Neither diet nor TUDCA changed renal medullary apoptosis in ETB def rats suggesting that medullary and cortical apoptosis may be mediated via distinct pathways. In conclusion, loss of functional ETB receptors leads to exaggerated renal cortical tubular injury and apoptosis. These findings indicate a high salt-induced reno-protective role for ETB receptor activation that may be blood pressure-independent. Supported by NIH T32 DK007545 to CDM and P01 HL95499 to DMP and JSP. 3.6 MEDULLARY HISTONE DEACETYLASE ENZYMES ARE CRITICAL FOR WATER BALANCE DURING HIGH SALT FEEDING Kelly Hyndman, Joshua Speed, Chunhua Jin, David M. Pollock, and Jennifer S. Pollock Med./Nephrology, Univ. of Alabama at Birmingham, 1720 2nd Ave. S, Kaul 830, Birmingham, AL, 35294. Histone deacetylases (HDAC) play a pivotal role in modifying lysines of histone and nonhistone proteins, thereby regulating transcription and protein function. We determined that a high salt diet increases expression of HDAC1 in the rat renal medulla. Moreover, overexpression of HDAC1 in collecting duct cells increases nitric oxide synthase-1, and decreases vasopressin V2 receptor expression. Thus, we hypothesized that HDAC1 functions as a pro-natriuretic/diuretic factor during high salt feeding. To test this hypothesis, male uninephrectomized Sprague Dawley rats were implanted with an iPrecio perfusion pump to facilitate interstitial infusion into the medulla of the remaining kidney. Pumps infused vehicle (33% DMSO in saline, N = 5) or the HDAC inhibitor, MS275 (1 mg/kg/day, N =10). Rats were given 4.0% NaCl diet for 7 days. Food and water intake were similar between controls and MS275 infused rats. However, urinary osmolality was significantly increased in MS275 infused rats (1441.8± 86 vs 1001 ± 64 mOsm/kg H2O). MS275 infusion led to a significant 19.2 ± 4.0 g increase in body mass compared to controls (3.4 ± 1.4 g). Sodium excretion was similar between the groups. Urinary nitrite/nitrate excretion was significantly reduced in MS275 rats compared to vehicle (4.3 ± 0.8 vs 8.6 ± 2.6 μmol/day, P < 0.05), while urinary ET-1 excretion was similar (3.4± 0.6 vs 5.0 ± 1.7 pg/day, P > 0.05). These data suggest that inhibition of Class 1 HDACs are critical for medullary nitric oxide production and regulation of water homeostasis. 3.7 RENAL ENDOTHELIN AND PURINERGIC SYSTEMS CONTRIBUTE TO SEXUAL DIMORPHISM IN SODIUM EXCRETION Eman Y. Gohar, and David M. Pollock 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 15 Div. of Nephrology, Dept. of Med., Univ. of Alabama at Birmingham, 834 KAUL, 720 20th St. S., Birmingham, AL, 35233. Renal endothelin-1 (ET-1) and purinergic systems are important regulators of Na homeostasis and may account for sex differences in cardiovascular and renal function. A link between these two systems has been recently demonstrated in vitro, however, the in vivo interaction is not clear. Therefore, we tested the hypotheses that (1) Na loading has sexually dimorphic effects on renal ET-1 production/release, and (2) purinergic signaling is involved in the renal ET-1 dependent response to a Na load. Our results showed that female Sprague Dawley rats on a normal Na diet had a 2.5fold higher ET-1 excretion than males (14.2 ± 3.0 vs. 5.6 ± 1.0 pg/day/kg, p<0.05). Urinary ET-1 increased 2-fold in males with increasing dietary Na (11.2 ± 0.8 vs. 5.6 ± 1.0 pg/day/kg, p<0.05), but remained unchanged in females, although diuresis and natriuresis were more robust in females compared with males. Furthermore, in males only, renal intramedullary infusion of suramin (purinergic (P2) receptor blocker) significantly blunted the increase in Na excretion and inner medullary ET-1 gene expression induced by intramedullary Na loading. In contrast, ET-1 gene expression in females did not change with intramedullary Na in the presence or absence of suramin. These data indicate that an activation of inner medullary purinergic (P2) and ET-1 signaling systems could play a more important role in the natriuretic response to Na loading in male compared to female rats. These studies were funded by NIH grants P01 HL69999 and P01 HL95499. 3.8 ENDOTHELIN RECEPTOR ANTAGONIST PROTECTS AGAINST ISCHEMIA/REPERFUSION-INDUCED ACUTE KIDNEY INJURY IN MALE BUT NOT IN FEMALE RATS Ryosuke Tanaka, Mamoru Ohkita, and Yasuo Matsumura Lab. of Pathological & Molecular Pharmacology, Osaka Univ. of Pharmaceutical Sci., 4-20-1 Nasahara, Takatsuki, 569-1094, Japan. Endothelin (ET)-1/ETA receptor system has been shown to play an important role in the pathogenesis of ischemia/reperfusion-induced acute kidney injury (AKI) and we have reported that ABT-627, a selective ETA receptor antagonist, markedly attenuated AKI in male rats. On the other hand, sex differences in AKI have been established in humans and experimental animals, and there are consistent findings that females are more resistant to the renal injury than males. Müller et al. have shown that the expression of prepro-ET mRNA in kidneys subjected to ischemia was significantly higher in males and the administration of ETA receptor antagonist abolished the differences in survival between sexes (Kidney Int 2002; 62: 1364-1371). However, this area has not been extensively studied, and further studies are needed to confirm the role of ET-1/ETA receptor system in the sex differences of AKI. In the present study, we examined the protective effects of ABT-627 on AKI, using male and female Sprague-Dawley rats. AKI was achieved by clamping the left renal artery and vein for 45 minutes followed by reperfusion, 2 weeks after contralateral nephrectomy. Although renal function in both male and female vehicle-treated AKI rats significantly decreased 1 day after reperfusion, these renal dysfunction were more severe in male than in female rats. In comparison to female rats, males exhibited much more severe renal injury, characterized by proteinaceous casts in tubuli and tubular necrosis. Since female rats have very mild injury in the above experiment condition, it may not be enough to show the protective effect of ABT-627. Therefore, female rats were subjected to a longer ischemic period (60-minute ischemia) to make severe injury, which is comparable to 45-minute ischemia-induced kidney injury in males. Intravenous bolus injection of ABT-627 (1 mg/kg) 5 minutes before ischemia markedly attenuated AKI in males, but not in females. Furthermore, the sex difference in AKI was abolished by ovariectomy and ABT-627 administration attenuated AKI in ovariectomized female rats. These findings suggest that ET-1/ETA receptor system is contributive to the sex difference in the pathogenesis of AKI. 3.9 EFFECTS OF COMBINED ENDOTHELIN A RECEPTOR AND RENIN-ANGIOTENSIN SYSTEM BLOCKADE ON THE REGRESSION OF CHRONIC KIDNEY DISEASE IN 5/6 NEPHRECTOMIZED REN-2 TRANSGENIC RATS Vera Certíková Chabova, Lenka Sedlakova, Zuzana Huskova, Libor Kopkan, Petra Skaroupkova, Sarka Dolezelova, Lenka Cervenkova, Zdenka Vanourkova, Ludek Cervenka, and Ivana Vaneckova Dept. of Nephrology, 1st Med. Fac., Charles Univ., U Nemocnice 2, Praha 2, CZ15500, Czech Rep., Ctr. of Experimental Med., IKEM, Vídeňská 1958/9, Praha 4, CZ-14021, Czech Rep., Inst. of Physiology, Academy of Sci., Vídeňská 1083, Praha 4, CZ-14220, Czech Rep. Study objective: We tested the hypothesis whether combined renin-angiotensin system (RAS) and endothelin 1 (ETA) receptor blockade could be more effective in the slowing of progression of chronic kidney disease (CKD) with already established sings of renal injury than antihypertensive treatment based on isolated RAS inhibition in 5/6 nephrectomized (NX) Ren-2 transgenic hypertensive rats (TGR). Methods: In five groups of rats: sham-operated TGR; untreated 5/6 NX TGR; 5/6 NX TGR with dual RAS blockade (trandolapril and losartan); 5/6 NX TGR with combined treatment with RAS and ETA receptor blockade (atrasentan), 5/6 NX was done at the age of 6 weeks and the treatments were initiated 6 weeks after 5/6 NX. Albuminuria was determined at weeks 6, 8, 12, 16, 20, 30. In other groups, concentrations of ANG II and ET1 were evaluated in the kidneys after 2 weeks of treatments. Results: Mortality of untreated 5/6 NX TGR was 100% at 17 week compared to sham-operated rats with mortality 0% at 30 week. Mortality of 5/6 NX TGR with RAS blockade was 32 % and in group with RAS and ETA receptor blockade 37 % at the end of 30 week. In 5/6 NX TGR, both treatments reduced enhanced albuminuria, plasma and kidney ANG II and cortical ET-1 concentrations similarly. Conclusion: There is no additive effect of combined ETA receptor and RAS blockade on the regression of CKD in 5/6 NX TGR most likely due to efficient reduction of ET-1 in the renal cortex by the dual RAS inhibition. 3.10 THE ENDOTHELIN SYSTEM MEDIATES RENAL ENDOPLASMIC RETICULUM STRESS DEVELOPMENT Carmen De Miguel, William C. Hamrick, Janet L. Hobbs, Masashi Yanagisawa, David M. Pollock, and Jennifer S. Pollock Med./Nephrology, Univ. of Alabama at Birmingham, 720 20th St. S., Kaul 840, Birmingham, AL, 35233, Molecular Genetics, Univ. of Texas Southwestern Med. Ctr., 5323 Harry Hines Blvd., Dallas, TX, 75390, Intl. Inst. for Integrative Sleep Med., Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8575, Japan. Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the exact molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and renal injury. These studies aimed to determine the role of ET-1 in renal ER stress development. Vascular endothelial cell ET-1 knockout (VEET KO) and flox control mice were used to study the role of ET-1 in renal vascular ER stress development in response to the ER stressor, tunicamycin (TM; i.p.). ER stress marker expression and renal apoptosis were determined by qRT-PCR and TUNEL assay, respectively. In flox mice, TM significantly increased relative ER stress marker expression in renal vessels (saline vs. TM, n=6-9/group, p<0.05; GRP94: 1.0±0.3 vs. 16.5±6.6, ATF-6: 1.0±0.5 vs. 6.8±3.5, and CHOP: 1.4±0.4 vs. 12.5±1.9) and led to increased outer medullary, non-vascular apoptosis (saline vs. TM, n=5-6/group, p<0.05: 0.4±0.1 vs. 5.9±1.8 TUNELcells/field). Interestingly, TM failed to increase renal vascular ER stress or renal apoptosis in VEET KO mice. The role of ET-1 receptors in renal ER stress was assessed using ETB deficient (sl/sl) or transgenic control (TG) rats. TM similarly increased cortical ER stress in both rat genotypes. However, in the outer medulla, TM led to a 13 to 22 fold increase from baseline only in sl/sl rats for sXBP-1, GRP78 and CHOP (n=7-8/group). Pre-treatment of TG control rats with ABT-627 (ETA antagonist; 5mg/kg/day) for 1 week prior to TM injection significantly reduced the ER stress response to TM in cortex (7 to 50 fold decrease for GRP78, sXBP-1, CHOP and caspase-12; n=3-4/group) and medulla (7 to 25 fold decrease for GRP78, ATF-4, spliced XBP-1, CHOP and ATF-6; n=3-4/group), also inhibiting renal apoptosis. ABT-627 pre-treatment failed to reduce renal ER stress development and apoptosis in sl/sl rats, indicating that a functional ETB receptor is key for the anti-ER stress and anti-apoptosis actions of ABT-627. In conclusion, endothelial-derived ET-1 is critical for the development of TM-induced renal ER stress and apoptosis. ETA receptor activation induces renal ER stress genes and apoptosis, while ETB receptor activation has reno-protective effects. These results highlight the possibility of targeting the ET-1 system as a therapeutic approach against ER stressinduced kidney damage. Funded by NIH T32 DK007545 to CDM and P01 HL95499 and P01 HL69999 to DMP and JSP. 3.11 DATA-LISTED ENDOTHELIN RECEPTOR TYPE B (ETB) DEFICIENCY RESULTS IN GREATER BLOOD PRESSURE LEVELS DURING PREGNANCY AND IN RESPONSE TO PLACENTAL ISCHEMIA-INDUCED HYPERTENSION IN RATS Frank Spradley Physiology & Biophysics, Univ. of Mississippi Med. Ctr., 2500 N. State St., Jackson, MS, 39216. Preeclampsia (PE) is a pregnancy-specific disorder of new-onset hypertension during pregnancy that threatens the lives of both mother and fetus. The mechanisms mediating this hypertension are unclear, but studies have shown that it most likely originnates from placental ischemia. Indeed, placental ischemia/hypoxia induced by reduced uterine perfusion pressure (RUPP) in experimental animals stimulates the release of soluble factors into the maternal circulation where they cause vascular dysfunction and hypertension. Importantly, blockade of the vasoconstrictive endothelin type A receptor (ETA) abolishes RUPP-induced hypertension implicating this receptor in PE. Although it is has been reported that RUPP reduces ETB-mediated blunting of ET-1-induced vasoconstriction and ETB expression in small mesenteric arteries, direct evidence implicating ETB in blood pressure regulation during pregnancy or in response to placental ischemia is scarce. Thusly, we tested the hypothesis that 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 16 ETB deficiency would result in increased blood pressure levels during pregnancy and even greater levels in response to placental ischemia. At eighteen weeks old, ETB deficient (def) and transgenic control (Tg) timed-pregnant rats were generated. Rats remained either in the normal pregnant (NP) group or RUPP surgeries performed at gestational day 14 with assessment of mean arterial blood pressure (MAP, carotid catheter) and pregnancy weights at day 19. This resulted in 4 groups: NP Tg (N=5); RUPP TG (N=4); NP ETB def (N=4); and RUPP ETB def (N=4). MAP was greater in NP ETB def than NP Tg (117±9 vs. 75±1, P<0.05). MAP levels were increased by RUPP in Tg (100±2, P<0.05) and to even greater levels in ETB def (146±6, P<0.05). Fetal weights were similar between NP ETB def and Tg (1.99±0.09 vs. 2.05±0.06) but were reduced significantly in RUPP ETB def (1.53±0.13, P<0.05) vs. Tg (1.68±0.15). Placental weights were similar in NP ETB def and Tg (0.53±0.02 vs. 0.54±0.02), and RUPP reduced these weights only in Tg (0.47±0.03, P<0.05) not ETB def (0.58±0.01). Placental sufficiency (fetal weight divided by placental weight, a marker of placental function) was similar in both NP groups (3.79±0.26 vs. 3.86±0.19) whereas RUPP reduced this in ETB def (2.62±0.21, P<0.05) but not Tg (4.08±0.65). In conclusion, these data indicate that the ETB receptor is important for blood pressure regulation during pregnancy and blunts the hypertension and placental dysfunction found in PE. Funding: T32HL105324-01. 3.12 CIRCADIAN REGULATION OF RENAL ENDOTHELIN-1 Joshua Speed, and David M. Pollock Med./Nephrology, Univ. of Alabama at Birmingham, 1720 2nd Ave. S., Kaul 834, Birmingham, AL, 35294. Rats lacking endothelin type B receptor function (ETB def) have an exacerbated circadian blood pressure rhythm. Endothelin-1 (ET-1) promotes renal excretion of Na and our lab has recently shown that endothelin-1 (ET-1) facilitates the storage and clearance of Na by the skin interstitium, providing a buffer for Na when salt intake is elevated. The goal of the current study is to determine if lack of ETB impairs the ability of the skin to store Na, and if this impairment plays a role exacerbated circadian blood pressure rhythm in ETB def rats. Transgenic control (Tg con) or ETB def rats were maintained on normal (0.8% NaCl) or high salt diet (4% NaCl) and urine was collected in 12-hour intervals (active and inactive periods). In a separate group of animals, rats were euthanized at 4-hour intervals beginning at zeitgeber time 0 (lights on), and skin was taken for the measurement of Na and water content. In Tg con rats, urinary excretion of ET-1, an indication of renal production, was significantly higher during the active period vs. inactive period in rats on NS (3.6±1.1 vs. 0.8±0.2 pg/12hr respectively), an effect that was more pronounced in HS fed rats (9.2±4.1 vs. 1.6±0.3 pg/12hr respectively). There was no difference in active vs. inactive period ET-1 excretion in ETB def rats on NS (6.6±2.2 vs. 4.6±1.7 pg/12hr respectively) suggesting an altered circadian pattern of renal ET-1 production. Interestingly, the pattern was restored in ETB def rats fed HS (2.2±1.0 vs. 9.2±2.5 pg/12hr inactive vs. active). In addition, rats fed HS had an increase in skin Na:H2O compared to NS fed rats; however, there was no difference between genotypes. These data suggest that ET-1 control of blood pressure rhythms occurs mainly through activation of renal ETB receptors to promote Na excretion, and not through a reduction in the ability of skin to buffer Na. This research was supported by NIH grants P01 HL95499, P0169999, and T32DK007545. 3.13 ETA RECEPTOR BLOCKADE IMPROVES THE DIFFERENTIAL DIURNAL NATRIURETIC RESPONSE TO AN ACUTE SALT LOAD IN MALE AND FEMALE ETB DEFICIENT RATS Jermaine Johnston, Joshua Speed, Chunhua Jin, and David M. Pollock Med./Nephrology, Univ. of Alabama at Birmingham, Kaul 834, 720 20th St. S., Birmingham, AL 35233. We have previously shown that rats lacking ETB receptors in non-neuronal tissues (ETB def) have an impaired ability to handle an acute sodium load that is time-of-day dependent. Subsequent studies suggested that the attenuated natriuretic response was more evident in male compared to female ETB def rats. The loss of the ETB receptor not only causes salt-dependent hypertension, but also results in elevated plasma ET-1 and uncontested ETA dependent vasoconstriction that can reduce sodium excretion. Therefore, we hypothesized that ETA receptor blockade (ABT-627, 5mg/kg/day, po) would improve the attenuated natriuretic response to acute salt loading. Male and female ETB def rats and littermate controls were implanted with telemetry transmitters to monitor mean arterial pressure (MAP). After a recovery period of at least a week, baseline urine samples were collected in 12 hr light/dark intervals. Rats were then given a single 900μEq Na salt load (NaCl) in 1 mL H2O by oral gavage at the beginning of their active (7pm-7am, dark) or inactive (7am-7pm, light) period. Control rats of both sexes given ABT-627 did not show any change in the pattern of urinary sodium excretion and excreted the majority of the salt load during the first 12 hrs regardless of time of day similar to untreated rats. ETB def males treated with ABT-627 showed a significantly improved natriuretic response to a salt load given during the inactive period after the first 12 hrs (456 ± 74 treated, n=4; 50 ± 91μEq Na/12hr untreated, n=5; P<0.05). ETB def females treated with ABT-627 also showed an improved natriuretic response during the first 12 hrs of the inactive period as well, though this improvement was not statistically significant (474 ± 34 treated, n=4; 183 ± 44μEq Na/12hr untreated, n=6; NS). MAP was unchanged in response to salt loading although ABT-627 significantly reduced MAP throughout the entire treatment period in both male and female rats (P<0.05). These results show that ETA receptor activation contributes to the impaired natriuretic response to salt loading in both male and female rats but does not diminish the time–related sex difference in sodium handling. This research was supported in part by NIH grants: P01 HL95499, P01 HL69999, and T32 DK007545. 3.14 ENDOTHELIN-1 INCREASES GLOMERULAR PERMEABILITY IN SICKLE CELL MICE Malgorzata Kasztan, Chiao-Wang Sun, Tim M. Townes, and David M. Pollock Cardio-Renal Physiology & Med., Univ. of Alabama at Birmingham, KAUL 840, 1720 2nd Ave. S., Birmingham, AL, 35294-0024, Biochemistry & Molecular Biol., Univ. of Alabama at Birmingham, KAUL 840, 1720 2nd Ave. S., Birmingham, AL, 35294-0024. Sickle cell disease (SCD) extensively alters renal structure and function, leading to nephropathy manifested by increased permeability of filtration barrier and albuminuria/proteinuria. The endothelium-derived peptide, endothelin-1 (ET-1), with its powerful vasoconstrictor and pro-inflammatory effects mediated primarily through ETA receptors, is elevated in plasma and urine of SCD patients and may contribute to the development of sickle cell glomerulopathy. Therefore, the aim of the study was to determine whether ET-1 contributes to increased glomerular permeability to albumin in SCD and if ETA receptors blockade ameliorates glomerular damage. Furthermore, because our preliminary studies showed sex differences in the vasoconstrictor response to ET-1 in sickle cell mice the study was designed to determine if sex differences exist in this response. Experiments utilized 12 week old humanized sickle cell mice (HbSS) and genetic controls (HbAA) recently developed by the Townes’ lab. Ambrisentan (ETA antagonist), A-182086 (ETA/B antagonist) or vehicle was administrated via drinking water and the concentration adjusted daily according to the intake (10mg/kg/day) for 2 weeks. Glomeruli were isolated for direct permeability measurements as a volume response of glomerular capillaries to an oncopressive medium generated by defined concentrations of albumin. Urinary protein excretion was determined using Bradford colorimetric method. Urinary albumin excretion was measured using enzyme immunoassay kit (GenWay). Glomerular permeability to albumin (Palb) was significantly higher in glomeruli from sickle mice (both in males and females) than control mice (0.50±0.07 and 0.47±0.06 vs. 0.13±0.02 and 0.10±0.2, respectively). Ambrisentan treatment significantly reduced the elevated Palb in glomeruli from male (0.24±0.05 vs. 0.50±0.07) and female (0.20±0.03 vs. 0.47±0.06) HbSS mice. ETA/B receptors antagonism with A-182086 also significantly decreased the Palb in glomeruli from male (0.28±0.06 vs. 0.50±0.07) and female (0.24±0.03 vs. 0.47±0.06) HbSS mice. However, there was no effect on albumin or protein excretion after the treatment with both antagonists. Treatment with both antagonists did not alter Palb in HbAA mice. These data support the hypothesis that ET-1 may play an important role in the development of sickle cell nephropathy and support the use of chronic ETA antagonism as a prospective treatment for sickle cell nephropathy. This work was supported by the program project grant on The Role of Endothelin-1 in Sickle Cell Disease (U01 HL117684), and UABUSCD O’Brien Center (grant DK079337). 3.15 SELECTIVE ENDOTHELIN-A RECEPTOR ANTAGONISM PREVENTS THE PROGRESSION OF ACUTE KIDNEY INJURY TO CHRONIC KIDNEY DISEASE Rebecca Moorhouse, Alicja Czopek, Lea Guyonnet, Olivia Lenoir, Pierre-Louis Tharaux, David Webb, David Kluth, and Neeraj Dhaun Clinical Pharmacology Unit, Univ. of Edinburgh, Little France Crescent, Edinburgh, EH16 4TJ, UK, Vascular Biol., Ctr. de Res., INSERM PARCC, Rue Leblanc, Paris, 75015, France, Ctr. for Inflammation Res., Univ. of Edinburgh, Little France Crescent, Edinburgh, EH16 4TJ, UK. Introduction: Acute kidney injury (AKI) is very common and associated with significant morbidity and mortality. AKI often progresses to chronic kidney disease (CKD) and endothelin-1 (ET-1) contributes to this. We hypothesized that therapeutic administration of selective ETA receptor antagonism would protect from the transition of AKI to CKD. Methods: 28 FVB mice underwent prolonged (50min) unilateral ischemia-reperfusion injury (IRI) with 28 days recovery. 14 mice received daily selective ETA antagonism (sitaxentan) starting 24h after IRI. We assessed blood pressure (BP) via telemetry, vascular function, renal injury and measures of the ET system. Results: Systolic BP increased by ~5mmHg after IRI and was associated with vascular dysfunction in both resistance and conduit vessels. Sitaxentan partly 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 17 prevented both of these. At 28d after IRI kidney weight was reduced (-55%) and associated with significant macrophage infiltration and fibrosis compared to the contralateral control kidney. Mice treated with sitaxentan had normal kidney weight, reduced macrophage infiltration and less fibrosis: IRI kidney vs. control kidney vs. IRI kidney with sitaxentan: F4/80 stain/high power field: 2.5 vs. 0.2 vs. 0.8%; picosirius red stain/high power field: 8.6 vs. 0.48 vs. 3.1%. For both macrophage infiltration and fibrosis, p<0.05 for IRI vs. control and for IRI vs. IRI with sitaxentan, p=ns for control vs. IRI with sitaxentan. Furthermore, an up-regulation of both the ETA (28-fold) and ETB (2-fold) receptors as well as pre-pro-ET-1 (10-fold) mRNA was seen in both the cortex and medulla of the IRI kidney relative to control. Angiotensinogen and renin mRNA was unchanged. With sitaxentan treatment ETA/ETB receptor and pre-proET-1 mRNA remained similar to baseline levels. Finally, renal ET-1 production increased following IRI and this was prevented by ETA receptor antagonism (fractional excretion of ET-1: IRI vs. IRI with sitaxentan: 47 vs. 16%, p<0.05). Conclusions: In an in vivo model of AKI progressing to CKD, ETA receptor antagonism reduced BP and vascular dysfunction and prevented progression of renal injury and ET system activation after AKI. Therefore, selective ETA receptor antagonism offers a potentially novel therapy for AKI. Translational studies are now warranted. Funded by a British Heart Foundation PhD studentship (FS/11/7829328). 3.16 THE ROLE OF A RENAL ALDOSTERONE-ENDOTHELIN FEEDBACK SYSTEM IN TOTAL NA BALANCE AND MINERALOCORTICOID ESCAPE Charles Wingo, I. Jeanette Lynch, Amanda Welch, Michelle Gumz, Brian Cain, and Donald Kohan Med., Univ. of Florida, 1600 SW Archer Rd., PO Box 100224, Gainesville, FL, 32610, Res., North Florida/South Georgia Health Sys., 1601 SW Archer Rd., Gainesville, FL, 32608, Biochemistry, Univ. of Florida, 1600 SW Archer Rd., PO Box 100245, Gainesville, FL, 32610, Nephrology, Univ. of Utah Hlth. Sci. Ctr. and Salt Lake City VAMC, 1900 East 30 North, Salt Lake City, UT, 84132. Aldosterone increases blood pressure (BP) by stimulating sodium (Na) reabsorption in the collecting duct (CD) and, by negative feedback, stimulates CD endothelin-1 (ET-1) that acts to inhibit Na and water reabsorption. We have previously provided evidence for a renal aldosterone-endothelin feedback system. We tested the hypothesis that this system is necessary for proper Na balance and BP control by comparing the effect of a high NaCl diet (2% gelled diet with saline to drink) and mineralocorticoid stimulation on wild-type mice (WT) and CD-specific ET-1 knockout mice (CD ET-1 KO). Metabolic balance and radiotelemetric BP were measured before and after treatment with desoxycorticosterone pivalate (DOCP, 0.07mg/g, IM). CD ET-1 KO mice consumed more high NaCl diet and saline and had greater urine output than WT. WT mice did not increase fluid intake or retention, body weight (BW), or systolic BP until during DOCP treatment. In contrast, the CD ET-1 KO mice increased fluid retention, BW, and systolic BP on the high NaCl diet alone. DOCP further increased systolic BP in the CD ET-1 KO, which exhibited greater Na and water retention and BW gain than WT. Unlike WT, CD ET-1 KO mice failed to return to neutral Na balance during 19 days of DOCP administration (mineralocorticoid escape). Thus, the absence of CD ET-1 expression impairs renal response to Na loading, which results in abnormal fluid and electrolyte handling when challenged with a high NaCl diet and DOCP treatment. We conclude that CD expressed ET-1 functions as an essential element necessary for mineralocorticoid escape. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-82680 to BDC & CSW, NHLBI grant P01 HL095499 to DEK, NIH postdoctoral fellowship 2T32HL083810 to AKW, and by funds from the Department of Veterans Affairs to CSW. 3.17 HIGH SALT INTAKE INCREASES ET-1 MEDIATED NATRIURESIS AND DIURESIS VIA THE ETB RECEPTOR IN RATS Chunhua Jin, and David M. Pollock Cardio-Renal Physiology & Med., Div. of Nephrology, Univ. of Alabama at Birmingham, KAUL 840, 1720 2nd Ave. S., Birmingham, AL, 35294-0024. Endothelin 1 (ET-1) causes vasoconstriction (anti-natriuresis) through ETA receptors and promotes natriuresis via ETB within the renal medulla, specifically in the collecting ducts. Direct intramedullary infusion of ET-1 has no effect on renal sodium and water excretion but is associated with ETA-dependent reductions in medullary blood flow. We have data to suggest that chronic high salt (HS; 4%NaCl) intake reduces renal medullary expression of ETA receptors, therefore leading us to hypothesize that HS intake would increase the natriuretic and diuretic response to renal medullary infusion of ET-1. Male Sprague Dawley (SD) rats were fed a normal salt (NS; 0.4% NaCl) or HS diet for 7 days. Rats were anesthetized and a catheter implanted in the renal medulla for interstitial infusion along with a ureteral catheter for urine collection. An adjustable occlude was placed around the aorta proximal to the left renal artery to maintain renal perfusion pressure throughout the protocol. After equilibration and a 40 min control period, the urine flow (UV) and sodium excretion (UNaV) responses to medullary infusion of a low dose of the ETB receptor agonist, sarafotoxin S6c (0.15 μg/kg/h), or non-selective agonist ET-1 (0.45 μg/kg/h) were determined over two 40 min infusion periods. Intramedullary infusion of S6c markedly increased UV and UNaV in the high salt treated rats (UV: 5.0±0.5 to 28.3±3.4 μl/min; UNaV: 0.87±0.12 to 1.98±0.18 μmol/min), but not in the NS treated rats. In HS treated rats, intramedullary infusion of ET-1 (0.45 μg/kg/h) induced a significant natriuretic responses compared to NS treated rats. (HS: 0.90±0.21 to 1.50±0.23; NS: 0.40±0.08 to 0.69±0.17 μmol/min). We conclude that high salt intake enhances the diuretic and natriuretic effects of ETB receptor activation in vivo consistent with a role for the ETB receptor in maintaining fluid-electrolyte balance that is counterbalanced by ETA activity. This work was supported by the program project grant on the Endothelin Control of Renal Hemodynamic and Excretory Function (PO1 HL-95499). 3.18 ENDOTHELIN RECEPTOR ANTAGONISM IN SICKLE CELL NEPHROPATHY Olivia Lenoir, Nathalie Sabaa, Carole Henrique, Léa Guyonnet, Cécile Fligny, Vincent Audard, and Pierre-Louis Tharaux Paris Cardiovascular Ctr., PARCC, INSERM, 56 rue Leblanc, Paris, 75015, France, Nephrology & Transplantation Dept., Assistance Publique-Hôpitaux de Paris (APHP), Henri Mondor Hosp., Nephrology & Transplantation Dept., 51, avenue du Maréchal de Lattre de Tassigny, Créteil, 94010, France. Introduction: Sickle-cell disease (SCD) is characterized by chronic hemolysis and recurrent episodes of vaso-occlusive events that affect the microcirculation and lead to ischemic tissue injury with multi-organ dysfunction. Sickle cell nephropathy (SCN), a major mortality risk factor in SCD, is characterized by an early increase in glomerular filtration rate with subsequent progressive decline of renal function. Focal and segmental glomerulosclerosis (FSGS) and hypertrophied glomeruli with distended capillaries are the major hallmarks of glomerular lesions. We investigated the effects of chronic mixed ET receptor antagonism in a model of SCD-mediated FSGS. Methods & Results: We used SAD1 (SAD) Hbβsingle/single hemizygous mice on the C57BL/6J background. At 3 months of age SAD mice displayed little evidence of chronic renal damage but significant glomerulomegaly compared to controls (average glomerular section area: 1733±155 vs. 1540±70μm, p<0.01). Glomerulomegaly persisted, and was worse, at 6 months of age (average glomerular section area: 2372±207 vs. 1519±180μm, p<0.001). In addition, SAD mice had increased glomerulosclerosis than controls (SAD vs. WT: 21±9 vs. 10±8%, p<0.05). Masson trichrome and Marinot silver staining showed marked thickening of glomerular basement membranes, mesangiolysis and sclerosis with diminished podocyte density as observed with podoplanin and WT-1 immunostaining (p<0.01 vs. controls). Based on these data we treated SAD mice and controls aged 3 months with the mixed ET receptor antagonist bosentan for 9 months in a preventative study, and 6 months old SAD mice for 6 months in a therapeutic study. We assessed blood pressure, kidney structure and function after 6 and 9 months of continuous treatment. In the preventative study, 6 months of bosentan therapy was associated with ~4-fold less glomerulosclerosis compared to untreated SAD mice (22±8 vs. 86±4%, p<0.001). Additionally, there was an 80% reduction in mean glomerular surface area (p<0.05). In the therapeutic study, there was a significant reduction in glomerulosclerosis (p<0.01) and glomerulomegaly (p<0.01) compared to untreated mice but this was less effective than in the preventative study (p<0.05). Conclusions: ET receptor antagonism is a potentially useful preventative or therapeutic approach in SCN. Based on these data clinical trials are warranted. 3.19 ENDOTHELIAL-DERIVED ENDOTHELIN-1 CONTRIBUTES TO RENAL DYSFUNCTION AND MORTALITY IN SICKLE CELL MICE Brandon Fox, Jonathan Heimlich, Chiao-Wang Sun, Tim Townes, Masashi Yanagisawa, David M. Pollock, and Jennifer S. Pollock Cardio-Renal Physiology & Med., Div. of Nephrology, Dept. of Med., Univ. of Alabama at Birmingham, 1720 2nd Ave S., Birmingham, AL, 35294, Georgia Regents Univ., 1459 Laney Walker Blvd., Augusta, GA, 30912, Dept. of Biochemistry & Molecular Genetics, Univ. of Alabama at Birmingham, 1720 2nd Ave S., Birmingham, AL, 35294, Intl. Inst. for Integrative Sleep Med. (WPI-IIIS), Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8575, Japan. Sickle cell disease (SCD) is a common genetic hematologic disease that causes progressive multi-organ pathology including nephropathy beginning as early as childhood. Endothelin-1 (ET-1) is known to be elevated in the plasma and urine of SCD patients and mice. A growing body of evidence using ET receptor antagonists in SCD mice suggests that ET-1 plays a role in the pathophysiology of SCD. However, the cellular source of increased ET-1 production in SCD has yet to be determined. We hypothesized that endothelial-derived ET-1 contributes to renal dysfunction and mortality in SCD mice. Vascular endothelial-specific ET-1 knockout (VEETKO) mice and genotype controls (flox) underwent bone marrow transplantation (BMT) with marrow derived from humanized SCD mice (HbSS). This method resulted in the de2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 18 velopment of SCD in mice lacking endothelial ET-1 production (HbSSVEETKO) and mice with endothelial ET-1 production (HbSS-flox). Full induction of the sickle hemoglobin phenotype requires approximately 12 weeks. The HbSS-VEETKO group showed a significant survival advantage over the HbSSflox group (p=0.026) with all HbSS-VEETKO mice surviving and only 58% of HbSS-flox mice surviving at 18 weeks post-BMT. Dysfunctional urineconcentrating ability exists in SCD patients and mice, most likely, secondary to progressive renal injury. To determine whether endothelial-derived ET-1 participates in the SCD mediated loss of urine-concentrating ability, osmolality was measured in both spot urines and 24hour metabolic cage urine collections. At five weeks postBMT, prior to the onset of the SCD phenotype, there was no difference in spot urine osmolality between HbSS-flox and HbSS-VEETKO mice (3244±268 vs. 3096±165 mOsm/kg, p>0.05). Following the onset of the SCD phenotype, mortality in the HbSS-flox group left only a single mouse for the assessment 24-hour urine osmolality, and this mouse showed a low urine osmolality (930 mOsm/kg). However, when compared to HbSS mice, HbSS-VEETKO mice demonstrated preserved urine-concentrating ability as indicated by significantly higher 24-hour urine osmolality (584±11 vs. 2512±361 mOsm/kg, p=0.006). These data indicate that endothelial-derived ET-1 is a major mediator of SCD pathophysiology and that lack of endothelial ET-1 production is sufficient to prevent renal dysfunction and mortality in SCD mice. This work was supported by U01 HL117684 to DMP and JSP and T32 HL007918 to BMF. 3.20 IDENTIFICATION OF EDN1-AS: A NOVEL LONG, NONCODING RNA IN THE REGULATION OF ENDOTHELIN-1 Kristen Solocinski, Sarah Barilovits, Amanda Welch, Charles Wingo, Brian Cain, and Michelle Gumz Med., Univ. of Florida, 1600 SW Archer Rd., Gainesville, FL, 32610, Biochemistry & Molecular Biology, Univ. of Florida, 1600 SW Archer Rd., Gainesville, FL, 32610, Nephrology, NF/SG Vet. Hlth. Sys., 1601 SW Archer Rd., Gainesville, FL, 32608. Long, non-coding (lnc)RNAs regulate gene expression via diverse mechanisms and can either activate or silence genes. Endothelin-1 (ET-1) is a peptide hormone that contributes to blood pressure regulation in a tissue-specific manner. We have identified a novel lncRNA transcribed from the human ET-1 gene (EDN1) locus. This antisense lncRNA, designated EDN1-AS, was first identified in human bronchial epithelial (S9) cells. We have also detected expression of EDN1-AS in human umbilical vein endothelial (HUVEC) cells, human mammary epithelial (HMEC) cells, and whole human kidney. These samples were derived from males and females, thus, EDN1-AS appears to be expressed in both sexes. The EDN1-AS lncRNA was also shown to oscillate over the course of 24 hours in synchronized human renal proximal tubule cells (HK2), suggesting that its expression may be regulated by the circadian clock mechanism. The clock is made of four core proteins: Bmal1, CLOCK, Cry (homologs 1 and 2), and Per (homologs 1-3). Interestingly, Per1 inhibits ET-1 expression in the kidney and Per1 was detected at the EDN1-AS promoter in HK2 cells. Furthermore, we have recently identified a murine homolog of EDN1-AS. Preliminary data indicate that expression of mEDN1-AS is higher in mouse kidney medulla compared to the cortex. Together these data demonstrate the identification of a novel ET-1-related lncRNA expressed in mice and humans, detectable in several cell types in both sexes, and may represent a previously unknown mechanism for regulation of ET-1 expression. Funding: This work was supported by NIH DK085193 and DK098460, and the ASN Foundation for Kidney Research to MLG, 2T32HL083810 to KS, 2T32HL083810 to AKW, and NIH DK082680 to BDC and CSW. 3.21 ETA RECEPTOR ACTIVATION CONTRIBUTES TO T CELL INFILTRATION FOLLOWING RENAL ISCHEMIA-REPERFUSION INJURY Erika Boesen Cellular & Integrative Physiology, Univ. of Nebraska Med. Ctr., 985850 Nebraska Med. Ctr., DRC1-5005, Omaha, NE, 68198-5850. Endothelin-1 (ET-1) and the ETA receptor undergo rapid and sustained upregulation in the kidney following ischemia and reperfusion (IR) injury, but data on the longterm consequences of this on renal function are limited. Renal IR injury increases the risk of hypertension, and while both the endothelin and immune systems have been implicated in hypertension, the contribution of the endothelin system to the inflamematory response following renal IR injury is poorly understood. The current study tested whether ETA receptor activation mediates adhesion molecule and chemotactic protein expression, and subsequent T cell infiltration following renal IR injury. Male C57BL/6 mice were treated with the selective ETA receptor antagonist ABT-627 (10 mg/kg/d p.o.) or vehicle (drinking water), from 2 days prior to 45 min unilateral renal IR. Treatment continued during recovery and mice were sacrificed at 24 h or 10 days post-IR (n=5-8 per group). The concentration of monocyte chemoattractant protein (MCP)-1 was significantly increased in the renal cortex of the ischemic versus contralateral kidney at 24 h post-IR (10-12 fold; Pkidney<0.001), but there was no significant difference between vehicle and ABT-627-treated mice. A similar increase was seen in the ischemic outer medulla, with no difference between vehicle and ABT-627-treated mice (MCP-1: 47±3 and 45±5 pg/mg protein respectively P>0.05). The mRNA expression of the adhesion molecule E-Selectin was significantly increased in the cortex of the ischemic kidney in both groups at 24 h (Pkidney<0.01), but this was not significantly affected by ABT-627. At 10 days post-IR, CD3 T cell numbers were dramatically increased in the ischemic versus contralateral cortex and outer medulla of both groups (Pkidney<0.001). ETA blockade significantly blunted the rise in T cell number in the outer medulla (Pkidney*treatment<0.05; 250±60 cells/mm in ABT-627treated mice, 422±37 cells/mm in vehicle), but this effect did not reach statistical significance in the cortex (Pkidney*treatment=0.1; 333±64cells/mm in ABT-627-treated mice, 490±71cells/mm in vehicle). These data suggest that ET-1 acting via the ETA receptor contributes to T cell infiltration of the outer medulla post-IR injury. This may have important implications for long-term blood pressure control following acute kidney injury, an area which awaits further investigation. Funding: American Heart Association Scientist Development Grant 12SDG8960028. 3.22 EVALUATION OF ENDOTHELIN A RECEPTOR (ETA) BLOCKADE ON THE PROGRESSION OF RENAL INJURY IN VARIOUS MODELS OF METABOLIC DISORDERS WITH PRE-EXISTING RENAL DISEASE Kasi McPherson, Denesha Spires, Lateia Taylor, Ashley Szabo-Johnson, and Jan M. Williams Pharmacology & Toxicology, Univ. of Mississippi Med. Ctr., 2500 N. State St., Jackson, MS, 39216. The endothelin (ET) system has been shown to play an important role in the development of renal injury via the endothelin A (ETA) receptor. Therefore, the current study examined whether chronic ETA blockade with ABT-627 would prevent the progression of renal injury in various animals models of metabolic disorders with pre-existing renal disease. In our type-1 diabetic model of renal disease (the streptozotocin (STZ) treated Dahl salt-sensitive (SS) rat), proteinuria increased to 353±34 mg/day and the rats were then separated into two groups: (1) vehicle and (2) ETA antagonist, ABT-627 (5mg/kg/day). After 6 weeks of treatment, MAP (via telemetry) decreased by 20% (139±10 vs. 108±6 mmHg, respectively, n=5) and proteinuria was markedly reduced in ABT-627 treated STZ-SS rats versus vehicle treated rats (310±32 vs. 517±68 mg/day, respectively, n=8). The degree of glomerular injury and renal interstitial fibrosis was significantly reduced in the kidneys of ABT-627 treated STZ-SS rats compared to vehicle STZ-SS rats. Next, we determined whether treatment with ABT-627 for 6 weeks would be beneficial in a type-2 diabetic model (T2DN rat) with pre-existing renal injury. While chronic ETA blockade did not have an effect on arterial pressure or proteinuria in T2DN rats, ABT-627 prevented the rise in plasma creatinine (Pcr) levels (1.4±0.2 vs. 0.9±0.1 mg/dL, respectively, n=6). Since the contribution of ET during the progression of renal disease associated with non-diabetic obesity has not been thoroughly investigated, we examined whether ETA blockade would be beneficial in preventing the progression of renal injury associated with obesity in the obese leptin receptor mutant Dahl salt-sensitive (SSmutant) strain derived from Zinc-finger nucleases. After 9 weeks of treatment, MAP was reduced by 20% (215±8 vs. 173±6 mmHg, respectively, n=3-5). However, we did not observe any differences in proteinuria between the groups. Similar to T2DN rats, chronic ETA blockade attenuated the increase in Pcr (1.3±0.2 vs. 0.7±0.1 mg/dL). Interestingly, treatment with ABT-627 increased the survival rate of SSmutant rats (83%, 5 of 6 vs. 50%, 3 of 6). In conclusion, these data indicate that ETA blockade prevents the decline in renal function in animal models suffering from hypertension, diabetes and/or obesity with pre-existing renal disease. This research supported by NIGMS NIH P20GM104357 and AHA 12SDG9440034. 3.23 ENDOTHELIN B RECEPTOR AGONIST, IRL-1620, PROVIDES NEUROPROTECTION AND ENHANCES ANGIOGENESIS IN DIABETIC RATS WITH CEREBRAL ISCHEMIA Anil Gulati, Monica Husby, and Mary Leonard Chicago Coll. of Pharmacy, Midwestern Univ., 555 31st St., Downers Grove, IL, 60515, Chicago Coll. of Hlth. Sci., Midwestern Univ., 555 31st St., Downers Grove, IL, 60515. The incidence of Type 2 Diabetes Mellitus (T2DM) and its associated cardiovascular complications are on the rise. Endothelin B (ETB) receptor agonist, IRL-1620, has been shown to provide neuroprotection and enhance neurovascular repair following cerebral ischemia, however its efficacy in comorbid models of ischemia and T2DM is unknown. T2DM was induced in rats using a HFD/STZ protocol, followed by permanent occlusion of the right middle cerebral artery (MCAO). IRL-1620 (5μg/kg,IV) was administered at 4, 6, and 8 hr on days 0, 3, and 6 post-MCAO. Survival and neurological function for the IRL-1620 group were significantly higher in 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 19 both non-diabetic (ND) and diabetic (D) rats compared to vehicle. A significant reduction in infarct volume was seen with IRL-1620 in both ND (80.8%) and D (69.6%) rats. There were significantly more PECAM-positive vessels/30μm brain slice in the ND and D IRL-1620 rats (9.1±0.5; 9.0±0.4) versus vehicle (5.9±0.4; 7.1±0.4). Immunostaining revealed a significant increase in ETB expression for ND (p<0.0001) and D IRL-1620 rats (p=0.04), compared to vehicle. Co-localization of VEGFand PECAM-positive endothelial cells was significant higher in D IRL-1620 rats (15.5±1.1%) compared to vehicle (10.2±0.8%). Results indicate that IRL-1620 is effective in cerebral ischemia in rats with T2DM by significantly reducing infarct volume, improving neurological/motor function, and enhancing angiogenesis. This study was supported by Midwestern University. 3.24 THE APOPTOTIC PATHWAY MEDIATES THE NEUROPROTECTIVE EFFECT OF IRL-1620 IN A RAT MODEL OF FOCAL CEREBRAL ISCHEMIA Anil Gulati, Seema Briyal, Anupama Puppala, and Luu Thanh Chicago Coll. of Pharmacy, Midwestern Univ., 555 31st St., Downers Grove, IL, 60515, Chicago Coll. of Osteopathic Med., Midwestern Univ., 555 31st St., Downers Grove, IL, 60515. Previous studies have shown that IRL-1620 enhances neurovascular remodeling following cerebral ischemia. It is possible that IRL-1620 provides protection to neurons by inhibiting the apoptotic pathway. Following middle cerebral artery occlusion (MCAO), rats received 3 injections of vehicle or IRL-1620 (5μg/kg,IV) at 2, 4 and 6 hr. Behavioral evaluation confirmed the induction of stroke. Rats were sacrificed and brains processed to evaluate protein expression of apoptotic markers. All procedures were approved by Midwestern University IACUC. Rats treated with IRL1620 showed significant improvement in neurological and motor function tests compared to vehicle. In addition, there was a significant decrease in infarct volume in IRL1620 treated rats (24.47±4.37mm) versus vehicle group (153.23±32.18mm). Antiapoptotic protein Bcl-2 expression was decreased and pro-apoptotic protein Bax expression was increased in vehicle-treated compared to sham (p<0.0001). IRL-1620 treatment showed significantly (p<0.01) increased expression of Bcl-2 and decreased expression of Bax. There were no changes in total Akt expression in sham, vehicle and IRL-1620 treated rats, however, there was an increase in pAkt in IRL-1620 treated rats compared to vehicle group (P<0.05) post-occlusion. The results demonstrate that IRL-1620 is a neuroprotective agent and attenuates the neuronal damage following cerebral ischemia in rats by preventing apoptosis. Study was supported by Midwestern University. 3.25 NEUROPROTECTIVE EFFECT OF APILIMOD IN ISCHEMIA REPERFUSION INJURY IN RATS Sunita Tiwari, Dinesh Tripathi, and Arti Verma Physiology, King George’s Med. Univ., Lucknow, 226003, India. Background: Cerebral stroke is a neurodegenerative disease and it is a major cause of death and disability throughout the world. Mitochondrial reactive oxygen species (ROS) generation and Lipid peroxidation level is increase during ischemia, which is responsible for neuronal death in cerebral stroke. Higher levels of ROS cause state of oxidative stress. Reactive oxygen species damage the DNA and play an important role in apoptosis. Lipid peroxidation is a well-established mechanism of cellular injury in animals, and refers to the oxidative degradation of lipids. So, Lipid peroxidation is an important factor in the pathophysiology of ischemia. Purpose: The aim of this study was to investigate the neuroprotective effects of apilimod (IL-12 inhibitor) on middle cerebral artery occlusion model of stroke in rats. Methods: Focal cerebral ischemia was induced in male S.D. rats (250 ± 20g) by occlusion of middle cerebral artery Apilimod was administered prior and post induction of ischemia to assess its therapeutic window. Neurological deficit were determined by Longa's score. Lipid peroxidation levels were evaluated by malondialdehyde assay and Glutathione (GSH) level was evaluated by 5,5-dithiobis-2-nitrobenzoic acid (DTNB) which is readily reduced by sulfhydryls forming a yellow substance which is measured spectrophotometrically at 412 nm. Result: Apilimod significantly ameliorated the neurological deficit. The biomarker of oxidative stress malondialdehyde (MDA) was also found to be significantly reduced following apilimod administration and increased the level of the antioxidant enzyme glutathione (GSH). Conclusion: These results show that apilimod has a preventive effect against ischemic stroke in animal model. Acknowledgement: We thanks the Defence Research & Development Organisation (DRDO), New Delhi, India for providing a grant (no. ERIP/ER/1103953M/01/1361). References: Longa EZ, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats.Stroke 20:84–91. Colado, M. I., O'Shea, E., Granados, R., Misra, A., Murray, T. K., and Green, A. R (1997) A study of the neurotoxic effect of MDMA ('ecstasy') on 5-HT neurones in the brains of mothers and neonates following administration of the drug during pregnancy. Br J Pharmacol 121:827-833. Anderson, M. E. (1985) Determination of glutathione and glutathione disulfide in biological samples. Methods Enzymol 113:548-555. 3.26 NEUROPROTECTIVE POTENTIAL OF ENDOTHELIN ETA RECEPTOR ANTAGONIST IN CEREBRAL ISCHEMIA MODELS Shyam Sharma, Tushar Deshpande, and Anil Gulati Pharmacology & Toxicology, Natl. Inst. of Pharma. Edu. and Res. (NIPER), Sector 67, S.A.S. Nagar (Mohali), 160062, India, Dept. of Pharma. Sci., Midwestern Univ., 555 31st St., Downers Grove, IL, 60515. Stroke is the second most common cause of death and the leading cause of disability, worldwide. The pathophysiology of stroke involves a complex multifactorial cascades. There is increasing evidence of involvement of endothelin in the development and progression of ischemic stroke. Therefore, endothelin is considered as one of the target for the treatment of cerebral ischemia-reperfusion injury. In this study we have investigated the effect of ETA receptor antagonist in two models viz. rat model of focal cerebral ischemia and gerbil model of global cerebral ischemia. Effect of endothelin receptor antagonist was assessed on cerebral blood flow, neurological score, neurological damage and biochemical changes after induction of cerebral ischemia. Middle cerebral artery occlusion (MCAO) resulted in drastic decrease (80%) in ipsilateral cerebral blood flow as measured by Laser Doppler flowmetry. Contralateral blood flow remained unaltered. Significant neurological deficits and damage was observed with MCAO. BMS 182874 showed neuroprotection in both animal models. BMS 182874 treatment significantly reduced ischaemic damage in focal cerebral ischemia model. This was associated with reduction in TNF α levels and MPO activity. BMS 182874 treatment (5 mg/kg) in gerbil model significantly decreased the locomotor activity as compared with control group. The response latency in passive avoidance test was also increased. This improvement in neurological parameters was well reflected in histopathology of brain. CA1 region of hippocampus was examined for number of survived neurons. BMS 182874 treatment increased the number of survived cells in CA1 region. Results of this study suggested the potential of pharmacological interventions targeted at endothelin receptors in cerebral ischemia models. 3.27 P66 SHC REGULATES ET-1-MEDIATED INTRACELLULAR CALCIUM HANDLING IN RENAL RESISTANCE ARTERIES AND CONTRIBUTES TO RENAL GLOMERULAR INJURY IN HYPERTENSION Oleg Palygin, Bradley Miller, Andrew Chong, Alexander Staruschenko, and Andrey Sorokin Physiology, Med. Coll. of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI, 53226, Med., Med. Coll. of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI, 53226. The pathogenesis of hypertension-induced nephropathy is associated with increased renal vascular resistance and loss of vascular responsiveness. Even though microvascular injuries are detected in the majority of patients with hypertension and are among causes of diseases leading to end stage renal disease, the pathophysiological mechanisms mediating renal microvascular dysfunction remain unknown. Excessive signaling via adaptor protein p66 Shc is likely to be one of the mechanisms of oxidative stress-related pathologies and the purpose of this study was to assess the contribution of p66 Shc to regulation of vascular responsiveness associated with hypertension-induced nephropathy. Dahl salt-sensitive (SS) rats exhibit many traits associated with salt-sensitive hypertension in man and became a well-established model for the study of salt-sensitive hypertension and accompanying cardiovascular disorders. By combining the precise modification of rat Shc1 gene with in vivo knock-in strategy we have generated a panel of mutant rat strains on the genetic background of SS rats. Endothelin-1 (ET-1), an important player in hypertension-related kidney diseases, induces the phosphorylation of p66 Shc serine 36 residue which is essential for p66 Shc to promote oxidative stress-related pathologies. ET-1-mediated elevation of intracellular Ca is strongly linked to renal microvascular contraction and is crucial for ET-1-induced contraction of smooth muscle cells. We used two-photon imaging of intracellular Ca handling in renal resistance arteries isolated either from wild type or from p66 Shc rat knockouts to investigate the role of p66 Shc in ET-1-mediated calcium signaling. We here report that overexpression of p66 Shc, observed in SS rats maintained on high-salt diet, partially impairs ET-1-mediated intracellular calcium handling in renal resistance arteries. ET-1-treated renal microvessels isolated from p66 Shc knockout rats demonstrate an increased elevation of intracellular calcium concentration when compared to renal vessels isolated from wild type SS rats. Since glomerular damage is one of direct consequences of renal vascular dysfunction, we have carried out glomerular injury scoring on PAS-stained paraffin sections. The comparison of glomerular damage in p66 Shc knockout and wild type SS animals revealed the mitigation of glomerular damage in the absence of p66 Shc. Our data suggest that p66 Shc knockout restores the calcium handling by ET-1 in smooth muscle cells of 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 20 isolated rat renal microvessels and mitigates renal damage in rats with hypertensioninduced nephropathy. 3.28 THE DOMINANCE OF RENIN-ANGIOTENSIN SYSTEM BLOCKADE OVER ENDOTHELIN RECEPTOR A BLOCKADE IN LOWERING OF BLOOD PRESSURE IN HETEROZYGOUS REN-2 TRANSGENIC RATS Ivana Vaneckova, and Josef Zicha Dept. of Hypertension, Inst. of Physiology AS CR, Videnska 1083, Prague 4, 14220, Czech Rep. Objective: We tested the hypothesis whether the addition of ETA blockade to reninangiotensin system (RAS) blockade would have other effects on principal vasoactive systems contributing to blood pressure (BP) maintenance in Ren-2 transgenic rats (TGR). Design and Methods: 5-week-old TGR rats were given either atrasentan, or a combination of atrasentan with angiotensin receptor blocker losartan, angiotensin converting enzyme inhibitor captopril, or direct renin inhibitor aliskiren for 4 weeks. At the end of the study, basal BP and acute BP responses to consecutive blockade of renin-angiotensin (RAS), sympathetic nervous (SNS), and nitric oxide (NO) systems were determined in conscious rats. Moreover, BP responses to acute inhibition of nifedipine-sensitive calcium influx through voltage-dependent calcium channels (LVDCC) were measured. Results: Atrasentan alone partially lowered BP, while in combination with all three RAS blockers BP was fully normalized. The BP lowering effects of all three RAS-blocking agents was dependent on the attenuation of both RASand SNS-dependent vasoconstriction. In all atrasentan-treated groups, NO-dependent vasodilation was substantially reduced and calcium influx through L-VDCC significantly decreased. Conclusion: Although the BP-lowering effects of combined ETA blockade and RAS blockade is predominantly dependent on the effects mediated by RAS blockade, further effects are attributable to ETA blockade. Grant 304/12/0259-Czech Science Foundation. 3.29 LINAGLIPTIN PROVIDES CEREBROVASCULAR PROTECTION VIA UPREGULATION OF ENDOTHELIAL ET-1 AND ETB RECEPTORS IN DIABETES. Mohammed Abdelsaid, Trevor Hardigan, and Adviye Ergul Physiology, Georgia Regents Univ., 1120 15th St. CA 2063, Augusta, GA, 30912, Charlie Norwood Vet., VA Med. Ctr., 1 Freedom Way, Augusta, GA, 30904, Program in Clinical & Experimental Therapeutics, Univ. of Georgia, 1120 15th St., CA 2063, Augusta, GA, 30912. We have shown that glycemic control with metformin or endothelin-1 (ET-1) inhibition with bosentan prevents AND restores diabetes-mediated pathological remodeling and neovascularization of the cerebrovasculature. Our recent data suggest that linagliptin, a member of the dipeptidyl peptidase-4 (DPP-4) inhibitor class of oral hypoglycemic agents, prevents cerebrovascular remodeling and dysfunction independent of its blood glucose lowering effects. We hypothesized that linagliptin provides cerebrovascular protection via modulation of the ET-1 system. Methods: 24 week old diabetic (Hemoglobin A1C >6.5%) and nondiabetic Wistar rats were treated for 4 weeks with either vehicle chow or chow containing 166mg/kg linagliptin. Retinal acellular capillary formation was measures as a surrogate marker for pathological neovascularization in diabetes. Brain microvascular endothelial cells (BMVEC) isolated from control or diabetic rats were also treated with (100 nM) linagliptin or Bosentan (10 uM) and tested for angiogenic properties with cell migration and tube formation assays. Expression of ET-1 and ET-B receptor were assessed using ELISA, RT-PCR and immunoblotting after treatment of BMVEC with linagliptin. Results: Linagliptin treatment significantly decreased retinal acellular capillaries in diabetes. BMVEC from diabetic animals showed a significant reduction in ET-B receptor. Linagliptin significantly increased ET-1 mRNA expression in both control and diabetic BMVEC and increased ET-B receptor mRNA and protein levels only in BMVECs from diabetic animals. In addition, linagliptin normalized the augmented angiogenic properties of diabetic BMVEC, such effect was blocked with the dual ET-A/Et-B antagonist, bosentan. Conclusions: Our results suggest that linagliptin-mediated increases in ET-1 and ETB provide cerebrovascular protection in a feed-forward mechanism. 3.30 ENDOTEHLIN-1: A FINAL COMMON PATHWAY LINKING PLACENTAL ISCHEMIA TO ENDOTHELIAL DYSFUNCTION AND HYPERTENSION DURING PREECLAMPSIA Joey Granger Physiology, Univ. of Mississippi Med. Ctr., 2500 N. State St., Jackson, MS, 39216. Despite being one of the leading causes of maternal death and a major contributor of maternal and perinatal morbidity, the mechanisms responsible for the pathogenesis of preeclampsia (PE) has yet to be fully elucidated. Growing evidence supports the concept that the placenta plays a central role in the pathogenesis of PE and that reduced uteroplacental perfusion leads to release of soluble placental factors such as the antiangiogenuc factor (sFlt-1) and tumor necrosis factor (TNF-α) and increased formation of the agonistic autoantibody to the angiotensin II type 1 receptor (AT1AA).There is growing evidence to suggest an important role for endothelin-1 (ET-1) in the pathophysiology of preeclampsia. Multiple studies have examined circulating levels of ET-1 and found elevated levels of plasma ET-1 in the preeclamptic group, with some studies indicating that the level of circulating ET-1 correlates with the severity of the disease symptoms. ET-1, however, is produced locally and plasma levels typically do not reflect tissue levels of the peptide. Animal studies from our lab has shown that a number of experimental models of preeclampsia (placental ischemia, sFlt-1 infusion, TNF-α infusion, and AT1-AA infusion) are associated with elevated tissue levels of ET-1. Moreover, we have reported that hypertension in pregnant rats, induced by placental ischemia or chronic infusion of sFlt-1, TNF-α, or AT1-AA can be completely attenuated by ETA receptor antagonism, strongly suggests that ET1 appears to be a final common pathway linking factors produced during placental ischemia to elevations in blood pressure. The findings that antagonism of the endothelin-A receptor is beneficial in numerous animal models of PE suggest that the ET system may be an intriguing target for pharmacological intervention in PE. 3.31 INDUCTION OF LONG-TERM ENDOTHELIN-1 OVEREXPRESSION CAUSES BLOOD PRESSURE RISE AND SMALL ARTERY STIFFENING Pierre Paradis, Suellen C. Coelho, Sofiane Ouerd, Júlio C. Fraulob-Aquino, Stefan Offermanns, and Ernesto L. Schiffrin Vascular & Hypertension Res. Unit, Lady Davis Inst. for Med. Res., Juif Genl. Hosp., McGill Univ., 3755 Chemin de la Côte Sainte-Catherine, Montreal, QC, J6K 5G3, Canada, Pharmacology, Max-Planck-Inst. for Heart & Lung Res., Ludwigstr. 43, 61231, Bad Nauheim, Germany. Background: The mechanisms of blood pressure (BP) regulation by endothelin (ET)-1 produced by endothelial cells are complex and remain unclear. Recently, we developed a transgenic mouse with tamoxifen-inducible endothelium-restricted human ET-1 overexpression (ieET-1) using Cre/loxP technology. ieET-1 mice exhibited BP rise after three weeks of induction in an ET type A receptor-dependent manner, in absence of vascular and kidney injury. It is unknown whether long-term exposure to ET-1 overexpression results in elevated BP elevation and vascular injury. Methods: Nine to 12-week old male ieET-1 mice and control ieCre mice expressing a tamoxifen-inducible Cre recombinase (CreER) under the control of EC-specific Tie2 promoter, were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and studied 3 months later. Metabolic cages were used to collect 24-hour urine for sodium, potassium and protein measurements. Renal artery flow (RAF) was assessed by ultrasonography. BP was determined by telemetry, plasma aldosterone by ELISA, and small mesenteric artery (MA) endothelial function and vascular remodeling by pressurized myography. Results: Systolic BP was increased in ieET-1 compared with ieCre mice (144±5 vs 117±3 mmHg, P<0.001). RAF was decreased in ieET-1 compared with control (1.9±0.2 vs 3.0±0.3 mL/min, P<0.01). The excretion of urinary sodium, potassium and protein was similar in both groups. Plasma aldosterone levels were increased in ieET-1 compared with ieCre mice (1.99±0.20 vs 1.29±0.12 ng/mL, P<0.05). MA endothelium-dependent relaxation responses to acetylcholine were impaired in ieET-1 compared to ieCre mice (36.3±4.7 vs 71.4±9.7%, P<0.01), whereas endothelium-independent relaxation responses to sodium nitroprusside were unchanged. MA media/lumen and media cross-sectional area were similar in both groups, but stiffness was increased in ieET-1 compared to ieCre mice, as indicated by leftward displacement of the stress-strain curves (strain at 140mmHg: 0.61±0.04 vs 0.71±0.02, P<0.05). Conclusions: The results demonstrate that long-term exposure to endothelial ET-1 overexpression caused sustained BP rise and small artery stiffening. This work was supported by Canadian Institutes of Health Research (CIHR) grants 37917 and 102606, a Canada Research Chair on Hypertension and Vascular Research and by the Canadian Fund for Innovation, all to ELS, and by a fellowship to SCC and to JCFA from the program of CsF/CNPq (Brazil). 3.32 ENDOTHELIN-1 OVEREXPRESSION EXAGGERATES TYPE 1 DIABETES-INDUCED ENDOTHELIAL DYSFUNCTION BY ALTERING OXIDATIVE STRESS BALANCE Pierre Paradis, Noureddine Idris-Khodja, Sofiane Ouerd, Muhammad Oneeb Rehman Mian, Jordan Gornitsky, Tlili Barhoumi, and Ernesto L. Schiffrin Vascular & Hypertension Res. Unit, Lady Davis Inst. for Med. Res., Juif Genl. Hosp., McGill Univ., 3755 Chemin de la Côte Sainte-Catherine, Montreal, QC, J6K 5G3, Canada. Objective: Increased endothelin (ET)-1 expression has been shown to cause endothelial dysfunction and oxidative stress. Plasma ET-1 is increased in patients with diabetes mellitus. Since endothelial dysfunction often precedes vascular complications in diabetes, we sought to determine whether ET-1 contributes to diabetes-induced endo2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 21 thelial dysfunction. We hypothesized that overexpression of ET-1 in the endothelium will exaggerate diabetes-induced endothelial dysfunction. Method: Diabetes was induced by streptozotocin treatment (STZ, 55 mg/kg/day, ip) for 5 days in 6 week old male wild-type (WT) mice and in mice overexpressing human ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Blood glucose, plasma ET-1 levels, mesenteric artery (MA) reactivity, mitochondrial superoxide production in aorta and endothelial nitric oxide synthase (Nos3), superoxide dismutase 1 (Sod1) and 2 (Sod2) mRNA expression in MA were determined. Results: STZ-induced diabetes was confirmed by increased glycemia in WT (P<0.001). Plasma ET-1 was increased in vehicle(15.9±4.6 vs 0.6±0.04 pg/mL, P<0.05) and STZ-treated eET-1 (4.9±0.6 vs 0.8±0.1 pg/mL, P<0.05) compared to respective WT controls. Diabetes caused a 27% reduction in vasodilatory responses to acetylcholine in WT (P<0.05), which was further decreased by 20% in eET-1 (P<0.05). Mitochondrial superoxide production was increased 1.8-fold by diabetes in WT (P<0.05) and further augmented by 31% in eET-1 (P<0.05). Nos3 expression was increased by 43% in vehicle-treated eET-1 compared to WT (P<0.05). Diabetes reduced Nos3 expression in eET-1 by 31% (P<0.05) but not in WT. Diabetes caused an increase in Sod1 (52%, P<0.05) and Sod2 (32%, P<0.05) expression in WT but not in eET-1. Conclusions: Increased levels of ET-1 exaggerate diabetes-induced endothelial dysfunction. This may be caused by a decrease in Nos3 expression, an increase in mitochondrial oxidative stress and a decrease in antioxidant capacity. This work was supported by Canadian Institutes of Health Research (CIHR) grants 37917 and 102606, a Canada Research Chair on Hypertension and Vascular Research and by the Canadian Fund for Innovation, all to ELS, and by fellowships to NIK (Fonds de recherché en santé du Québec), SO (CIHR), MORM (Canadian Vascular Network and Lady Davis Institute/TD Bank studentship) and TL (Richard and Edith Strauss Postdoctoral Fellowship). 3.33 ENDOTHELIN-1 OVEREXPRESSION PRESERVES ENDOTHELIAL FUNCTION IN MICE WITH VASCULAR SMOOTH MUSCLE CELL-RESTRICTED PPARΓ KNOCKOUT Pierre Paradis, Noureddine Idris-Khodja, Sofiane Ouerd, Michelle Trindade, Jordan Gornitsky, Asia Rehman, Tlili Barhoumi, Stefan Offermanns, Frank J. Gonzalez, and Ernesto L. Schiffrin Vascular & Hypertension Res. Unit, Lady Davis Inst. for Med. Res., Juif Genl. Hosp., McGill Univ., 3755 Chemin de la Côte Sainte-Catherine, Montreal, QC, J6K 5G3, Canada, Pharmacology, Max-Planck-Inst. for Heart & Lung Res., Ludwigstr. 43, 61231, Bad Nauheim, Germany, Ctr. for Cancer Res., Natl. Cancer Inst., Bldg. 37, Rm. 3106, Bethesda, MD, 20892. Objective: Peroxisome proliferator-activated receptor γ (PPARγ) agonists reduce blood pressure (BP) and vascular injury in hypertensive rodents and humans. Pparγ inactivation in vascular smooth muscle cells (VSMC) using a tamoxifen inducible Cre-Lox system enhanced angiotensin II-induced vascular injury. Transgenic mice overexpressing endothelin (ET)-1 selectively in the endothelium (eET-1) exhibit endothelial dysfunction, increased oxidative stress and inflammation. We hypothesized that inactivation of Ppar in VSMC (smPparγ) will exaggerate ET-1-induced vascular damage. Methods and Results: Eleven week-old male control, eET-1, smPparγ and eET-1/smPparγ mice were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and sacrificed 4 weeks later. Systolic BP was 14 mmHg higher in eET-1 compared to control (P<0.05) and unaffected by Pparγ inactivation. Mesenteric artery (MA) vasodilatory responses to acetylcholine were reduced by 37% in smPparγ (P<0.05) compared to control. Reactive oxygen species levels were increased in eET-1 (70%), smPparγ (120%) and eET-1/smPparγ (180%) compared to control (P<0.05). MA monocyte chemoattractant protein-1 expression was 70% higher in smPparγ compared to control (P<0.05), and unaffected by ET-1 overexpression. Perivascular fat monocyte/macrophage infiltration was >2-fold higher in eET-1 and smPparγ compared to control (P<0.05), and further increased by 68% in eET-1/smPparγ (P<0.05). Nitric oxide synthase (Nos) 3 mRNA expression was increased by 21% in eET-1 compared to WT (P<0.05). Nos2 expression was increased 3.7 and 2-fold in eET-1 and smPparγ compared to WT, respectively (P<0.05). The Ednra/Ednrb mRNA ratio was decreased by 29% in eET-1/smPparγ compared to smPparγ (P<0.05).Conclusion: Increased ET-1 paradoxically preserves endothelial function in mice with smPparγ inactivation, despite enhanced oxidative stress and inflammation.This work was supported by Canadian Institutes of Health Research (CIHR) grants 37917 and 102606, a Canada Research Chair on Hypertension and Vascular Research and by the Canadian Fund for Innovation, all to ELS, and by fellowships to NIK (Fonds de recherche en santé du Québec), SO (CIHR), and TL (Richard and Edith Strauss Postdoctoral Fellowship). 3.34 ROLE OF THE MYELOID ENDOTHELIN-B RECEPTOR IN ANGIOTENSIN II MEDIATED END-ORGAN DAMAGE Lea Guyonnet, Neeraj Dhaun, Philippe Bonnin, Veronique Baudrie, Rebecca Moorhouse, Alicja Czopek, Olivia Lenoir, David Webb, David Kluth, and PierreLouis Tharaux Vascular Biology, Ctr. de Res., INSERM PARCC, Rue Leblanc, Paris, 75015, France, Clinical Pharmacology Unit, Univ. of Edinburgh, Little France Crescent, Edinburgh, EH16 4TJ, UK, Ctr. for Inflammation Res., Univ. of Edinburgh, Little France Crescent, Edinburgh, EH16 4TJ, UK. Introduction: Hypertension is common and in the majority of cases its cause remains unknown. Recent interest has focused on the role of macrophages (Mφ) in blood pressure (BP) regulation. Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor mediating its effects through two receptors – the endothelin-A receptor (ETA) and endothelin-B (ETB) receptor. The ETB receptor has a specific role in ET-1 clearance. We investigated the role of the Mφ ETB receptor in a model of angiotensin II (Ang II)-mediated end-organ damage. Methods: Mφ ETB receptor deficient mice (LysMETB) and controls were exposed to Ang II infusion for 6 weeks under a high salt diet. We assessed BP via telemetry, cardiac structure and function and endothelial function by Doppler ultrasound, end-organ injury and plasma and urine ET-1. Results: At baseline, components of BP did not differ between groups and increased similarly with Ang II. Whereas after 6 weeks of Ang II LysMETB and controls had similar left ventricular hypertrophy and cardiac insufficiency, endothelial function was better in LysMETB at both baseline and after Ang II (% dilation of basilar artery in response to CO2, LysMETB vs. controls: baseline: 20 vs.11%, p<0.01; at 6 weeks: 11 vs.0%, p<0.01). Baseline renal function and proteinuria did not differ between groups. After Ang II, LysMETB showed similar renal function compared to controls but less proteinuria (urine albumin:creat, mg/mmol: 208 ± 10 vs. 530 ± 25, p<0.01), glomerulosclerosis (34 ± 2 vs. 61 ± 4%, p<0.001), and fewer renal Mφ compared to controls (F4/80 staining per high power field, LysMETB vs. controls: 1.1 ± 0.7 vs.3.2 ± 0.5%, p=0.02), although similar levels of CD3 T cells. Plasma ET-1 was no different at baseline but increased more in LysMETB with Ang II (LysMETB vs. controls after 6 weeks Ang II: 3.7 ± 0.7 vs.1.4 ± 0.2 pg/ml, p=0.03). Urine ET-1 was similar baseline and 6 weeks. Discussion: Deletion of the Mφ ETBR is associated with a blunting of the effects of systemic Ang II infusion as reflected by less endothelial dysfunction, reduced inflammation and end-organ damage. The mechanisms for these effects are the focus of ongoing research. Partly funded by a British Heart Foundation Intermediate Clinical Research Fellowship (FS/13/30/29994). 3.35 HIGH DIETARY FAT INTAKE IS ASSOCIATED WITH ENHANCED ENDOTHELIN-1 VASOCONSTRICTOR TONE Caitlin Dow, Jared Greiner, Nicole Schuette, Brian Stauffer, and Christopher DeSouza Integrative Physiology, Univ. of Colorado, Boulder, Clare Small Rm. 114, 1725 Pleasant St., Boulder, CO, 80309, Dept. of Med., Univ. of Colorado, Denver, 12605 E. 16th Ave., Aurora, CO, 80045. High dietary fat intake is associated with increased cardiovascular disease (CVD) risk. Endothelin (ET)-1 is a potent vasoconstrictor peptide synthesized and released by the endothelium that contributes to CVD risk. We hypothesized that high dietary fat intake is associated with enhanced ET-1 system activity. Fifty sedentary, non-obese adults (41-71 years) were studied: 30 (21M/9F) who habitually consumed a lower fat diet (LFD; 30±5% total calories from fat) and 20 (12M/8F) who habitually consumed a high fat diet (HFD; 40±5% total calories from fat). Dietary fat classifications were based on American Heart Association’s recommendation to consume <35% of total calories from fat and were determined via 4-day diet records. Forearm blood flow (FBF; plethysmography) was determined in response to intra-arterial infusions of ET1 (5 pmol/min for 20 min), a selective ETA receptor blockade (BQ-123, 100 nmol/min for 60 min), and non-selective ETA/B receptor blockade (BQ-123+BQ-788 [50 nmol/min for 60 min]). The vasoconstrictor response to ET-1 was significantly blunted (~60%; P<0.05) in the HFD compared with the LFD group. ETA receptor blockade resulted in greater vasodilation (~40%; P<0.05) in the HFD (19%) versus LFD (11%) group. There was no additional dilator response to non-selective ETA/B blockade in either group. These results indicate that habitual consumption of a diet high in fat is associated with enhanced ETA-mediated ET-1 vasoconstrictor tone. 3.36 VITAMIN C SUPPLEMENTATION REDUCES ET-1 SYSTEM ACTIVITY IN OVERWEIGHT AND OBESE ADULTS Caitlin Dow, Jared Greiner, Danielle Templeton, Brian Stauffer, and Christopher DeSouza Integrative Physiology, Univ. of Colorado, Boulder, Clare Small Rm. 114, 1725 Pleasant St., Boulder, CO, 80309, Dept. of Med., Univ. of Colorado, Denver, 12605 E. 16th Ave., Aurora, CO, 80045. Endothelin (ET)-1 system activity is elevated in overweight and obese (OW/OB) adults, contributing to vasomotor dysregulation and increased vascular risk. Regular aerobic exercise is a lifestyle strategy that reduces ET-1-mediated vasoconstrictor tone; however, >50% of OW/OB adults do not exercise. Vitamin C supplementation beneficially influences endothelial function. We determined whether daily vitamin C supplementation is as efficacious as regular aerobic exercise in lowering ET-1 system activity in OW/OB adults. 35 sedentary, OW/OB adults completed three months of 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 22 either vitamin C (500 mg/day, timed-release) supplementation (VC; n=20; 15M/5F; 58±2 yr; BMI: 31.4±0.6 kg/m) or aerobic (walking) exercise training (EX; n=15; 10/5 57±2 yr; 29.3±0.7 kg/m). Forearm blood flow (FBF; plethysmography) responses to intra-arterial infusion of ET-1 (5 pmol/min for 20 min) and selective ETA receptor blockade (BQ-123, 100 nmol/min for 60 min) were determined before and after intervention. There were no anthropometric changes in response to either VC or EX. Vasoconstriction to ET-1 increased similarly (~2-fold; P<0.05) in response to both interventions. Prior to intervention, resting FBF to BQ-123 was significantly increased (~20%; P<0.05) in both groups. Similar to EX, after VC supplementation, BQ-123 did not elicit a significant change in resting FBF. Vitamin C supplementation represents an effective lifestyle strategy for reducing ET-1-mediated vasoconstrictor tone in OW/OB adults. 3.37 BORDERLINE-HIGH TRIGLYCERIDES AND ENDOTHELIN1 VASOCONSTRICTOR TONE Caitlin Dow, Jared Greiner, Kyle Diehl, Brian Stauffer, and Christopher DeSouza Integrative Physiology, Univ. of Colorado, Boulder, Clare Small Rm. 114, 1725 Pleasant St., Boulder, CO, 80309, Dept. of Med., Univ. of Colorado, Denver, 12605 E. 16th Ave., Aurora, CO, 80045. Borderline high fasting plasma triglyceride (TG) concentrations (150-199 mg/dL) are an independent risk factor for cardiovascular (CV) disease risk, though the mechanisms underlying this risk are unclear. Endothelin-1 (ET-1) is a potent endogenous vasoconstrictor synthesized and released by the endothelium. Enhanced ET-1 vasoconstrictor tone is linked to CV pathologies. We hypothesized that ET-1 system activity is elevated in adults with borderline-high plasma TG concentrations compared with those with normal plasma concentrations. Eighteen sedentary, overweight adults (43-70 years) were studied: 9 (7M/2F, BMI: 28.0±1.2 kg/m) with normal plasma TG concentrations (56-120 mg/dL); and 9 (7M/2F; BMI: 27.1±1.0 kg/m) with borderline-high plasma TG concentrations (150-192 mg/dL). Forearm blood flow (FBF; plethysmography) was determined in response to intra-arterial infusions of ET1 (5 pmol/min for 20 min) and a selective ETA receptor blocker (BQ-123, 100 nmol/min for 60 min). Vasoconstriction to ET-1 was ~10-fold lower (P<0.05) in the borderline-high TG compared with the normal TG group. In response to BQ-123, FBF increased ~25% in the borderline-high TG versus ~10% in the normal TG group (P<0.05). These results indicate that borderline-high TG concentrations are associated with increased ET-1 mediated vasoconstrictor tone. Enhanced ET-1 system activity may contribute to the increased cardiovascular burden associated with elevations in TG concentrations. 3.38 C-REACTIVE PROTEIN DOES NOT INFLUENCE ENDOTHELIN-1 SYSTEM ACTIVITY IN HEALTHY ADULTS Caitlin Dow, Jared Greiner, Grace Lincenberg, Brian Stauffer, and Christopher DeSouza Integrative Physiology, Univ. of Colorado, Boulder, Clare Small Rm. 114, 1725 Pleasant St., Boulder, CO, 80309, Dept. of Med., Univ. of Colorado, Denver, 12605 E. 16th Ave., Aurora, CO, 80045. C-reactive protein (CRP) is an inflammatory cytokine that has been shown to be an independent predictor of future atherothrombotic events. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide synthesized and released from the endothelium. In addition to its role in vasoregulation, ET-1 hyper-bioactivity is atherogenic. ET-1 is sensitive to inflammatory stimuli; however, the influence of CRP on ET-1 system activity is unknown. We tested the hypothesis that ET-1-mediated vasoconstrictor tone is enhanced in adults with elevated plasma CRP concentrations. Sixty non-obese, sedentary adults (42-70 years) were studied: 20 (13M/7F; BMI: 26.5±0.5 kg/m) with CRP <1.0 mg/dL (low CRP; 0.5±0.1 mg/dL); 20 (13M/7F; BMI: 26.4±0.6 kg/m2) with CRP 1.0-3.0 mg/dL (moderate CRP; 2.0±0.1 mg/dL); and 20 (13M/7F; BMI: 27.9±0.8 kg/m2) with CRP >3.0 mg/dL (high CRP; 6.3±0.5 mg/dL). Forearm blood flow (FBF; plethysmography) was determined in response to intra-arterial infusions of ET-1 (5 pmol/min for 20 min) and selective ETA receptor blockade (BQ-123, 100 nmol/min for 60 min). Vasoconstriction to exogenous ET-1 was not significantly different (P>0.05) between the low (10%), moderate (11%) and high (7%) CRP groups. FBF response to BQ-123 was almost identical between groups; all groups demonstrated a marginal (~10%) but significant vasodilator response. These results indicate that ET-1 system activity is not influenced by elevations in CRP. 3.39 ENDOTHELIN-1 STIMULATES ENDOTHELIAL-DERIVED MICROPARTICLE RELEASE Philip J. Kavlich, Tyler D. Bammert, Jamie G. Hijmans, Kyle J. Diehl, Grace M. Lincenberg, Ryan T. Fay, Whitney N. Riaekvam, Jared J. Greiner, and Christopher A. DeSouza Integrative Physiology, Univ. of Colorado, Boulder, 354 UCB, Boulder, CO, 80309. Endothelial microparticles (EMPs) are vesicles shed from the endothelium and are a marker of endothelial injury. Elevated circulating EMPs are associated with several cardiovascular, inflammatory and metabolic diseases. Endothelin (ET)-1, a potent vasoconstrictor peptide, is associated with endothelial damage and atherogenesis. We tested the hypothesis that ET-1 increases the release of EMPs from arterial endothelial cells. In three separate experimental units, human aortic endothelial cells (HAECs) were grown to confluence; thereafter, cells were incubated for 24 hours in the absence and presence of ET-1 (100 and 200 pmol) and the selective ETB receptor antagonist, BQ-788 (1μmol). Following incubation, media was collected, centrifuged (200 x g: 10 min). Prior to analysis, samples were centrifuged (13,000 x g: 2 min) and 100μL of the supernatant was collected and microparticles were labeled with anti-human CD31, a marker expressed on microparticles derived from endothelial cells, and CD42b, a non-specific surface marker. Samples were analyzed using flow cytometry and EMPs were defined as CD31+/CD42bevents less than 1.0 μm. EMP release into the media significantly increased from 8 MP/μL under basal conditions to 16 and 18 MP/μL with ET-1 stimulation at 100 and 200 pmol, respectively. The presence of BQ-788 blunted (~35%) ET-1 stimulated EMP release at both concentrations. Furthermore, boiled ET-1 had no effect on EMP release indicating that the response to ET-1 did not represent a general protein effect. These results indicate that: 1) ET-1 induces EMP release from endothelial cells; and 2) this effect is mediated, in large part, via an ETB receptor process. 3.40 EARLY-LIFE STRESS INDUCES EPIGENETIC REGULATION OF THE ET SYSTEM IN ADULT MALE MICE Dao Ho, Mariah Burch, David M. Pollock, Jennifer S. Pollock Cardio-Renal Physiology & Med., Div. of Nephrology, Univ. of Alabama at Birmingham, Kaul Bldg., 1720 2nd Ave S., Birmingham, AL, 35294. Early-life stress (ELS) is a risk factor for cardiovascular diseases associated with a dysfunctional endothelin (ET) system; however, little is known of whether ELS epigenetically alters the ET system. We hypothesized that ELS induces vascular dysfunction and a dysregulated ET system in an epigenetic manner. Using adult male control mice (CON) and mice exposed to maternal separation with early weaning (MSEW), a model of ELS, we assessed the gene expression of 84 different chromatin remodeling enzymes and components of the endothelin system (ET-1, ETA and ETB receptor) in thoracic aorta, as well as ex vivo vascular function by wire myography. Mean arterial pressure, heart rate and body weight were not different between groups (n=6). However, plasma ET-1 level was elevated in MSEW compared to CON (1.5 vs 1.2 pg/ml, n=8, p=0.05). Aortic mRNA expression of histone deacetylase (HDAC) 1, 6 and 9 were significantly elevated in MSEW compared to CON (RTqPCR; 1.17, 1.29 and 1.67 fold increase from CON, respectively, n=3, p<0.05). Western blot demonstrated that only HDAC9 protein levels were significantly elevated in aortas of MSEW (1.88 fold increase from CON, n=6, p=0.01). MSEW displayed aortic endothelial dysfunction when compared to CON (i.e., blunted acetylcholine relaxation; 67.6 vs 89.9% maximal relaxation, n=10, p=0.01) that was reversed by ex vivo treatment with pan-HDAC inhibitor, trichostatin A (TSA). MSEW had increased aortic ETB receptor mRNA expression (2.2 fold change from CON, n=6, p<0.05), which was reduced by ex vivo TSA treatment. Expression of ET-1 and ETA receptor were not different between groups. To assess the mechanism of MSEW-induced ETB receptor expression, we tested the hypothesis that HDAC9 overexpression in cultured mouse aortic endothelial cells (MAECs) increases ETB receptor expression. Interestingly, overexpression of GFP-tagged HDAC9 via plasmid transfection in MAECs did not increase ETB receptor mRNA expression when compared to vector-transfected control, suggesting that MSEW-induced ETB receptor expression may be localized in non-endothelial cells. We propose that MSEW may induce HDAC9-regulated ETB receptor expression in aorta as a compensatory response to pathological postnatal programming of vascular function. In conclusion, ELS induces HDAC-mediated endothelial dysfunction and regulation of the endothelin system, possibly contributing to the programming of increased cardiovascular disease risk. F32 HL116145; P01 HL69999. 3.41 TREATMENT WITH DPPIV INHIBITOR LINAGLIPTIN REDUCES PLASMA ET-1 AND ET-1-INDUCED CEREBROVASCULAR HYPER-REACTIVITY IN DIABETES Trevor Hardigan, Yasir Abdul, and Adviye Ergul Physiology, Georgia Regents Univ., 1120 15th St., Augusta, GA, 30904, Physiology, Charlie Norwood Vet. Affairs Med. Ctr., 950 15th St., Augusta, GA, 30904. Objective: Diabetes is associated with macrovascular and microvascular complications leading to cerebrovascular disease. We have previously shown that endothelin-1 (ET-1) contributes to cerebrovascular dysfunction and remodeling in the Goto-Kakizaki (GK) rat model of type 2 diabetes. We also reported that glycemic control with metformin prevented vascular changes and decreased plasma ET-1 levels in this model. Since metformin has direct antioxidant effects in addition to its insulin sensitizing actions, discerning whether the beneficial effects were due to glycemic control or due to direct effects of metformin remained unknown. Thus, we 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 23 used linagliptin (LINA), a member of the dipeptidyl peptidase-IV (DPP-IV) inhibitor class of glucose lowering agents, to further investigate the impact of glycemic control on ET-1 activation and vascular dysfunction in diabetes. We hypothesized that glycemic control with LINA treatment would decrease blood glucose levels, plasma levels of ET-1, and ameliorate the increase in ET-1-induced vascular hyperreactivity in the GK rat. Methods and Results: Male diabetic GK (HA1C%>6.5%) and Wistar rats (age 24 weeks) were fed either normal or LINA chow for 4 weeks at a concentration of 166 mg/kg of chow (n=5-6/group). Plasma was collected following treatment, and basilar arteries were mounted on a wire myograph where a dose response curve to ET-1 (10-10 M) was performed. LINA treatment did not lower blood glucose in GK rats (HA1C%: GK: 8.02±0.27 vs. GK LINA: 8.20±0.33) but decreased plasma ET-1 levels (pg/ml) in diabetic GK rats (Wistar: 1.22±0.02, Wistar LINA: 1.21±0.02, GK: 1.85±0.02***, GK LINA: 1.75±0.01, ***=p< 0.0001 vs. Wistar, =p<0.01 vs. GK). LINA treatment decreased ET-1-induced contraction in basilar arteries from diabetic rats: (Area Under the Curve: Wistar: 562.2±111.1, Wistar LINA: 410.8±63.34, GK: 815.1± 62.65**, GK LINA: 548.7±34.86, **=p<0.01 vs. Wistar, =p< 0.05 vs. GK). Conclusions: Contrary to our hypothesis, linagliptin did not decrease blood glucose levels in the GK rats. This allowed us to examine the effects of linagliptin on the ET-1 system independent of glycemic control. We show here for the first time an effect of linagliptin on plasma ET-1 levels and ET1 vascular hyper-reactivity in GK rats. DPP-IV inhibition with linagliptin holds potential as a possible therapy for diabetic vascular disease due to its reduction of ET-1 levels and consequent vasoprotection. 3.42 HIGH GLUCOSE-MEDIATED INCREASE IN PERINUCLEAR ETA AND ETB EXPRESSION IN HUMAN BRAIN VASCULAR SMOOTH MUSCLE CELLS IS NOT AMELIORATED BY LINAGLIPTIN Yasir Abdul, Trevor Hardigan, and Adviye Ergul Physiology, Georgia Regents Univ., 1120 15th St., Augusta, GA, 30912, Physiology, Charlie Norwood VA Med. Ctr., 950 15th St., Augusta, GA, 30904. Introduction: We have previously reported that endothelin-1 (ET-1) promotes cerebrovascular remodeling in diabetes via ETA and ETB receptor activation on vascular smooth muscle cells. Dipeptidyl peptidase-4 (DPP-4) inhibitors have emerged as a new class of anti-diabetic therapies. We recently observed that treatment with linagliptin (DPP-IV inhibitor) in type-2 diabetic Goto-Kakizaki (GK) rats prevents vascular remodeling and improve the contractile functions independent of its blood glucose lowering effect. Thus, the goal of the current study was to explore the effect of linagliptin on the brain vascular smooth muscle cell (BVSMC) ET system in an in vitro model. Hypothesis: Linagliptin treatment can prevent the high glucoseinduced increase in secretion of ET-1 and expression of ET receptors in BVSMCs. Methods: Serum starved human BVSMCs were subjected to either normal glucose (5.5mM ) or high glucose (25 mM) containing media and treated with the linagliptin (100nM) for 24 hours. Media was collected for measurement of ET-1 by ELISA and cell lysates were prepared for the measurement of ETA and ETB receptor expression. Cells were also cultured on glass slides and incubated with ETA and ETB receptor antibodies for the immunostaining. Results: Immunostaining showed a remarkable increase in expression of ETA receptor in peri-nuclear areas of high glucose treated cells, however there was no significant difference after linagliptin treatment. Similarly, ETB receptor expression was also increased in the high glucose treated cells but the linagliptin treatment did not change the expression. High glucose exposure increased (not significant) the expression of both ETA and ETB receptor in cell lysate of BVSMCs. The linagliptin treatment did not show any significant difference in the expression of both ETA and ETB receptors. The secretion of ET-1 measured in media was also not significantly different between the groups. Conclusions: The dose of linagliptin and the duration of exposure used in the present study did not show any significant effect of linagliptin on ET receptor system of BVSMCs. Thus, it is concluded that the attenuation of brain vascular remodeling by linagliptin could be attributed through the other cell types (like endothelial cells) of the vasculature and may be independent of the ET system. High glucose-induced perinuclear ET receptor localization needs to be further studied. 3.43 POTENTIAL ASSOCIATION OF CIRCULATORY LEVEL OF ENDOTHELIN-1 AND DIABETES IN RURAL WOMEN IN BANGLADESH Subrina Jesmin, Yujiro Matsuishi, Arifur Rahman, Majedul Islam, Nobutake Shimojo, Sayeeda Nusrat Sultana, Sohel Zaedi, Shila Akhtar, AKM Ahsan Habib, Osamu Okazaki, Naoto Yamaguchi, Takashi Miyauchi, Satoru Kawano, and Taro Mizutani Inst. of Clinical Med., Fac. of Med., Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba,Ibaraki, Japan, Dept. of Cardiology, Shaheed Ziaur Rahman Med. Coll., Bogra City, Bogra, Bangladesh, 3 (NCGM), Natl. Ctr. for Global Hlth. & Med., Shinjuku-ku, Tokyo, Japan, Ctr. for Hlth. Sci., Ibaraki Prefectural Univ., Ami, Ibaraki, Japan. Aims: Diabetes Mellitus (DM) is a global epidemic affecting approximately 285 million people and has a high (and rising) prevalence in both developed and, more recently, developing countries. DM has, in particular, become an important health concern in the South Asian region, with an estimated increase in the prevalence of diabetes of over 151% between the years 2000 and 2030. Endothelin-1 (ET-1), a potential marker of endothelial dysfunction has been shown to be elevated in diabetic subjects. However, to date, the circulatory profile of ET-1 and its association with diabetes mellitus (DM) have not been investigated in any South Asian country like in Bangladesh. The present study assessed circulating levels of ET-1 in subjects with or without DM and further examined the association of ET-1 with clinical and metabolic parameters in Bangladeshi rural women. Main Methods: A total of 2022 rural Bangladeshi women were studied using a cross-sectional survey. Then further analysis was done on a case control basis having DM (n=179) and non-DM (204). Multiple regressions were used to examine the association between circulatory ET-1 level and DM. We used the World Health Organization’s (WHO) STEPS approach (modified), health-related behaviour (step 1), basic physical measures (step 2) and basic biochemical investigations, such as levels of blood glucose and cholesterol (step 3). Key findings: DM prevalence in the current study was 9.1%. ET-1 levels were significantly increased in diabetes subjects [DM vs. non-DM: 3.11 ± 0.16 vs. 1.97 ± 0.03, p<0.001]. In multivariable analyses, after adjusting for age, ET-1 had significant positive association with waist circumference (p=0.029), fasting plasma glucose levels (p=0.001), and HDL-C (p=0.016). In multiple regression analysis considering ET-1 level as dependent variable, we found plasma glucose level and HDL-C is the independent determinants for plasma ET-1 levels in Bangladeshi rural women. Through tertile analysis, we found mean ET-1 levels significantly increase as levels of blood glucose increases (p for trend<0.001). Significance: A higher concentration of ET-1 among DM subjects suggests the possible endothelial dysfunction in this apparently healthy population. The relation of ET-1 and DM needs further investigations to define the clinical predictive value of plasma ET-1 levels in DM for the South Asian population. This work has been supported by Ministry of Education and Science in Japan. 3.44 AMELIORATION OF ACUTE LIVER INJURY WITH THE BLOCKADE OF PROTEASE ACTIVATED RECEPTOR (PAR)2 THROUGH THE SUPPRESSION OF UPREGULATED LEVELS OF ENDOTHELIN-1 AND TNF-Α IN A RAT MODEL OF ENDOTOXEMIA Subrina Jesmin, Sohel Zaedi, Nobutake Shimojo, Shila Akhtar, Arifur Rahman, Yujiro Matsuishi, Naoto Yamaguchi, Sayeeda Sultana, Satoshi Gando, Satoshi Sakai, Satoru Kawano, Taro Mizutani, and Takashi Miyauchi Dept. of Emergency and Critical Care Med., Univ. of Tsukuba, Tennodai 1-1-1, Tsukuba, 305-8575, Japan, Ctr. for Tsukuba Adv. Res. Alliance (TARA), Univ. of Tsukuba, Tennodai 1-1-1, Tsukuba, 305-8575, Japan, Dept. of Cardiovascular Med., Univ. of Tsukuba, Tennodai 1-1-1, Tsukuba, 305-8575, Japan. Sepsis is associated with tissue hypoperfusion and metabolic impairment, which may contribute to the subsequent development of multiple organ failure. The liver is one of the organs that are normally damaged during the pathogenesis of sepsis and septic shock. Numerous studies on infectionor sepsis-induced liver injury have been reported, however, to date no effective treatment has been reported on this disorder. Some studies suggested that circulating endothelin (ET)-1 is elevated in sepsis. Here, we examined the time-dependent alterations of ET-1, NO and inflammatory cytokine, such as TNF-α in liver tissue in a septic rat model. Normal Wistar rats were administered with lipopolysaccharide (LPS: 15 mg/kg) and sacrificed at different time points (1h, 3h, 6h and 10h). The classical features of acute liver injury, such as infiltration of inflammatory cells, hepatocytic necrosis, were seen in LPS administered rats, and plasma bilirubin, GOT and GPT levels were also significantly changed. A 28-fold increase in ET-1 level was observed in liver tissue at 10 h after LPS administration, while a peak increase of 14-fold ET-1 mRNA level was seen 1 hour after LPS administration in liver tissue. Levels of hepatic TNF-α peaked (4.5-fold) at 1 hour of sepsis. Endotoxemia often triggers exuberant inflammatory responses and activation of the coagulation cascade, and interactions between inflammation and coagulation may be important in this setting. Protease-activated receptors (PARs) connect coagulation proteases to cellular responses and represent one mechanism by which coagulation might affect inflammation. Of the 4 mammalian PARs, PAR1, PAR3, and PAR4 are activated by thrombin, and PAR2 can be activated by coagulation proteases VIIa and Xa but not thrombin. Interestingly, PAR2 blocking peptide improved the status of liver injury, an effect that was associated with suppression of TNF-α elevation, and normalization of ET-1. Conclusions: The present study revealed a distinct chronological expression of ET-1 in LPS-mediated liver injury and suggested that blockade of PAR2 played a crucial role in treating liver injury in the septic rats, via a 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 25 Laura Rosanò, Elisa Semprucci, Piera Tocci, Valentina Caprara, Roberta Cianfrocca, Rosanna Sestito, Valeriana Di Castro, Gabriella Ferrandina, and Anna Bagnato Experimental Res. Dept., Regina Elena Natl. Cancer Inst., Via Elio Chianesi, 53, Rome, 00144, Italy, Gynecologic Oncology Unit, Catholic Univ. of Rome, Largo Agostino Gemelli, 8, Rome, Italy. The endothelin-1 (ET-1)/ET A receptor (ETAR) signalling pathway is a wellestablished driver of epithelial ovarian cancer (EOC) progression. One key process promoted by ET-1 stimulation is tumor cell invasion, which requires the scaffolding functions of β-arrestin-1 (β-arr1); however, the potential role of ET-1 in inducing invadopodia formation/function, which are crucial for cellular invasion and tumor metastasis, is completely unknown. We set out to molecularly dissect the ET-1 signaling pathway that control EOC cell invasion through regulation of small Rho GTPase family members. Results of this work revealed that ET-1/ETAR activates RhoA and RhoC GTPase, and downstrem ROCK1, in association with actin-based dynamic remodelling and enhanced cell invasion, that are inhibited by macitentan as well as by the ROCK inhibitor, Y-27632. Mechanically, we found that these effects are accomplished by the direct interaction of b-arrestin 1 (β-arr1) with PDZ-RhoGEF, a RhoA specific guanine nucleotide exchange factor, representing the predominant activator of RhoA and C downstream of ETAR. Interestingly, ETAR-mediated invasive properties are related to the regulation of invadopodia, as evaluated by colocalization of actin/cortactin with areas of matrix degradation, and activation of cofilin pathway, which is crucial for regulating invadopodia activity. Depletion of PDZ-RhoGEF, or β-arr1, or RhoC, as well as the treatment with macitentan, significantly impairs invadopodia function, matrix-metalloprotease activity, and invasion, demonstrating that β-arr1/PDZ-RhoGEF interaction mediates ETAR-driven ROCK-LIMK-cofilin pathway through the control of RhoC GTPase activity. In vivo, macitentan is able to inhibit metastatic dissemination and Rho GTPase expression. Finally, analysis of EOC human tissues reveals that ETAR overexpression, which is significantly associated with poor prognosis, positively correlates with RhoC expression. Overall, our results have uncovered a novel role for the complex βarr1/PDZ-RhoGEF as regulator of ET-1/ ETAR-induced motility and metastasis, unravelling ET-1 axis as regulator of invadopodia protrusions through RhoC/ROCK/LIMK/cofilin pathway during the initial steps of EOC invasion. 3.50 CLINICAL USE OF SERUM BIG ENDOTHELIN-1 LEVELS AS A TUMOUR MARKER FOR HAEMANGIOSARCOMA Shinya Fukumoto, Kaname Saida, Taku Miyasho, Tsuyoshi Kadosawa, Hidetomo Iwano, and Tsuyoshi Uchide Vet. Internal Med. Dept. of Small Animal Clinical Sci., Rakuno Gakuen Univ., 582 Bunkyodai Midorimachi, Ebetsu, 069-8501, Japan, Inst. for Biological Resources and Functions, Natl. Inst. of Adv. Ind. Sci. and Tech., Umezono 1-1-1, Tsukuba, 3058566, Japan, Companion Animal Nutrition Dept. of Veterinary Sciences, Rakuno Gakuen Univ., 582 Bunkyodai Midorimachi, Ebetsu, 069-8501, Japan, Vet. Oncology Dept. of Small Animal Clinical Sci., Rakuno Gakuen Univ., 582 Bunkyodai Midorimachi, Ebetsu, 069-8501, Japan, Vet. Biochemistry Dept. of Basic Vet. Med., Rakuno Gakuen Univ., 582 Bunkyodai Midorimachi, Ebetsu, 069-8501, Japan. In veterinary, haemangiosarcoma (HSA) is an important malignant neoplasm of dogs that originates from vascular endothelial cells. HSA is the most common splenic malignant neoplasm in dogs, approximately 50% of splenic masses were HSA. This study explored the suitability of using serum big endothelin-1 (ET-1) as a tumour marker for canine spontaneous HSA. Serum big ET-1 was measured in dogs with splenic HSA (n = 23), splenic malignant tumours other than HSA (n = 10), benign splenic lesions (n = 12) and normal healthy dogs (n = 17) by ELISA. Serum big ET-1 levels in dogs with HSA were significantly (P <0.01) higher than in other dogs. High sensitivity (100%, 95% confidence interval 92-100%) and specificity (95%, 95% confidence interval 87-95%) for HSA diagnosis were obtained using a cut-off of 17 pg/mL according to receiver operating characteristic (ROC) curves (area under ROC curve 0.95). PPET1, ETA, VEGF and Hif1-α mRNA expression, measured by realtime PCR, were elevated in HSA compared to normal tissues. These findings suggest that elevated serum big ET-1 could be used as a diagnostic marker for canine HSA. 3.51 REGULATION OF THE CARDIAC ENDOTHELIN SYSTEM AND CARDIOMYOCYTE HYPERTROPHY BY GPER Matthias Meyer, Natalie Fredette, Christoph Daniel, Kerstin Amann, Matthias Barton, and Eric Prossnitz Dept. of Internal Med., Univ. of New Mexico Hlth. Sci. Ctr., 915 Camino de Salud NE, Albuquerque, NM, 87131, Dept. of Nephropathology, Friedrich-AlexanderUniv. Erlangen-Nürnberg, Krankenhausstrasse 8-10, Erlangen, 91054, Germany, Molecular Internal Med., Univ. of Zürich, LTK Y44 G22, Zürich, 8057, Switzerland. Aging is a major risk factor for cardiac hypertrophy that is partly mediated by increased activity of the local endothelin system. Endothelin-1 potently induces cardiomyocyte hypertrophy through stimulus-dependent activation of ETA or ETB receptors, which are involved in a complex cardiac signaling network of G proteincoupled receptors that control cardiomyocyte adaptation. Because signaling pathways of the G protein-coupled estrogen receptor (GPER) and endothelin receptors functionally interact in vascular smooth muscle, we hypothesized that GPER may also play a role in the age-dependent activation of the cardiac endothelin system. Using hearts from senescent (24 month-old) GPER-deficient and wild-type mice, we analyzed myocardial mRNA expression of prepro-endothelin-1, endothelin converting enzymes (ECE), as well as ETA and ETB receptors by RT-PCR. In addition, cardiomyocyte size was determined. In hearts of GPER-deficient mice, we found reduced expression of ECE-2 (2.3-fold, n=6, p<0.05 vs. wild-type mice), a rate-limiting enzyme in endothelin-1 synthesis. Furthermore, ETB receptor mRNA levels were decreased (1.7-fold, n=6, p<0.05 vs. wild-type mice), while ETA receptor or preproendothelin-1 expression was unaffected by GPER deletion. Consistent with lower ECE-2 and ETB receptor expression that may result in reduced myocardial endothelin-1 signaling, cardiomyocyte size was decreased 1.9-fold by GPER deletion (28±2 vs. 51±3 �m, n=6-8, p<0.0001 vs. wild-type mice). In conclusion, these findings indicate that endogenous GPER is partly required for activation of the endothelin system and cardiomyocyte hypertrophy during cardiac aging, suggesting a localized interaction between GPER, ECE-2, and endothelin receptor signaling that may be pharmacologically relevant to target age-dependent cardiac hypertrophy and resulting congestive heart failure. Supported by the National Institutes of Health (R01 CA127731 & CA 163890 to E.P.), the Swiss National Science Foundation (grants 135874 & 141501 to M.M. and grants 108258 & 122504 to M.B.), and the Interdisciplinary Center for Clinical Research (IZKF) Erlangen (project F1 to K.A.). N.F. was supported by NIH training grant HL07736. 3.52 ENDOTHELINS AS MARKERS OF CARDIOVASCULAR PROTECTION IN ADULTS WITH ISOLATED DEFICIENCY OF GROWTH HORMONE (IDGH) Sydney Leao, Carlos Aurélio Santos Aragão , Manuel Sena de Freitas, João Victor Lima Dantas, Williasmin Batista Souza, Sandro Leão Mattos, Hertaline Menezes do Nascimento, Manuel Hermínio Aguiar-Oliveira, Michael R. Dashwood, and Tania Maria de Andrade Rodrigues Dept. of Pathology, Fed. Univ. of São Paulo, Botucatu St., no. 740. Vila Clementino, São Paulo, 04038032, Brazil, Grp. of Molecular Anatomy, Dept. of Morphology, Fed. Univ. of Sergipe, Marechal Rondon Ave., b.b. Rosa Elze, São Cristóvão/SE, 49035-100, Brazil, Dept. of Med., Fed. Univ. of Sergipe, Claudio Batista St., b.b. Sanatorium Aracaju/SE, 49035-100, Brazil, Dept. of Clinical Biochemistry, Univ. Coll. London, Pond St., London, UK. Background: In the city of Itabaianinha, located on the southern state of Sergipe, Brazil, there is a group of 105 individuals affected by isolated GH deficiency (IDGH) due to a mutation of the GH receptor gene (GHRH). Previous studies in patients with GH deficiency acquired in adulthood demonstrated increased cardiovascular morbidity and mortality. However, the literature claims that patients with elevated serum Endothelins (ET) have Cardiovascular Risk (CVR) increased due to endothelial dysfunction caused by atherosclerosis. Objective: To evaluate the serum levels of ET in adults with IDGH and its correlation with CVR. Methods: It is a descriptive and prospective case-control study. A total of 34 patients, 20 patients with IDGH coming of Itabaianinha/SE and 14 healthy individuals residing in the city of Aracaju (state capital of Sergipe, Brazil.) comprised the control. They were submitted to dosage of serum ET through Elisa test. All experiments were conducted in accordance with the Declaration of Helsinki. Statistical analysis was performed by measures of central tendency and variance and comparisons between groups was performed by chi square test. The statistical significance level was less than 5% (p <0.05). Results: The control group was composed of seven men (50%) and seven women (50%) with mean age of 42.2 ± 19.9 years; while the dwarves sample consisted of 11 men (55%) and nine women (45%) with mean age of 46 ± 15.5 years. The mean values of the serum ET from controls were ET 70.78 ± 39.02 pg / ml and for patients with IDGH was 25.15 ± 6.40 pg/ml (p= 0.0003 CI: 92%). Conclusion: It is concluded that the ET levels in patients with IDGH were inferior to controls, so it appears that people with IDGH have less cardiovascular events than healthy patients. But further studies are needed to corroborate this claim. 3.53 KNOCKOUT OF ENDOTHELIN-1 IN VASCULAR ENDOTHELIAL CELLS AMELIORATES CARDIAC MITOCHONDRIA DYSFUNCTION AFTER MYOCARDIAL INFARCTION IN DIABETES TYPE 2 MICE Hary Sakti Muliawan, Ken Ichi Hirata, Kazuhiko Nakayama, Koji Ikeda, Keiko Yagi, and Noriaki Emoto Internal Med. Cardivascular Div., Kobe Univ., 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan, Clinical Pharmacy, Kobe Pharma. Univ., 4-19-1 Motoyama-Kitamachi, Higashinada Ward, Kobe, 658-0003, Japan. 2015 APS Conference 14 International Conference on Endothelin: Physiology, Pathophysiology and Therapeutics ABSTRACTS OF INVITED AND VOLUNTEERED PRESENTATIONS 26 Background: Persistent elevation of circulating endothelin-1 (ET-1) has been detected in diabetic patients and associates with low cardiac function post myocardial infarction. However, the role of ET-1 in linking diabetic heart and myocardial infarction is not well understood. Several studies suggest that ET-1 can induce mitochondria dysfunction which is an important feature of heart failure. Thus, we hypothesize that ET-1 might increase diabetic heart vulnerability to myocardial infarction through impairment of mitochondrial biogenesis and function. Methods: We induced diabetes mellitus type 2 in vascular endothelial cells specific-ET-1 knock out mice (VEETKO) and their wild type (WT) littermates using combination of streptozotocin injection and western diet. Six weeks after, we performed myocardial infarction using cryoinfarction procedure. Cardiac histology, function, gene expression, and mitochondrial biogenesis were evaluated one week after cryoinfarction. Results: Diabetic WT mice exhibited lower cardiac function, higher mitochondria structural abnormality, downregulation of PGC-1α/NRF1/OXPHOS signaling, higher ADP/ATP ratio, and higher oxidative stress compared to diabetic VEETKO mice. In vitro study revealed lower mitochondrial area, size, PGC-1α, and OXPHOS gene down-regulation in H9C2 cell treated with combination of high glucose and ET-1. Conclusion: These results show that vascular ET-1 increase diabetic heart vulnerability post myocardial infarction by aggravating mitochondrial biogenesis impairment. Reducing circulating ET-1 level might be beneficial to protect diabetic heart from myocardial infarction by preserving mitochondrial biogenesis and function. Support: Grant-in-Aid for Scientific Research (C) 26460213 and 18590813 from the Japan Society for the Promotion of Science. 3.54 ATTENUATION OF ENDOTHELIN-1-INDUCED CARDIOMYOCYTE HYPERTROPHY THROUGH ESTROGEN PRETREATMENT VIA NON-GENOMIC PATHWAY: POTENTIAL INVOLVEMENT WITH VEGF SYSTEM Nobutake Shimojo, Subrina Jesmin, Yujiro Matsuishi, Sohel Zaedi, Shila Akhtar, Arifur Rahman, Sayeeda Nusrat Sultana, Kazutaka Aonuma, Takashi Miyauchi, and Satoru Kawano Emergency & Critical Care Med., Fac. of Med., Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, Japan, Dept. of Cardiology, Shaheed Ziaur Rahman Med. Coll., Bogra City, Bogra, Bangladesh, Inst. of Clinical Med., Fac. of Med., Univ. of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, Japan. Estrogen (β-estradiol), the female hormone, has been reported to inhibit cardiac hypertrophy and apoptosis by different research groups in recent days. Thus, although the anti-hypertrophic action of estrogen in heart has begun to be appreciated, the potential mechanism underlying the estrogen-mediated cardioprotection is unknown. Endothelin (ET)-1, a potent vasoconstrictor, induces hypertrophic changes in neonatal cardiomyocytes at both morphological and molecular levels. Targeting ET-1 in the prevention of heart failure is crucial to suppress the cardiovascular diseases. Indeed, pathological cardiac hypertrophy progressively leads to heart failure. In the first part of the current study, we investigated whether estrogen confers beneficial effect against ET-1-induced cardiomyocyte hypertrophy and if cardioprotective, then whether this estrogen mediated cardiac anti-hypertrophic action is genomic or non-genomic. The doses of estrogen and ET-1 were optimized based on preliminary doseand time-dependent studies. At day 4 of culture, neonatal rat cardiomyocytes were divided into three groups: control, ET-1 (10nM) treated and estrogen-pre-treated (1μM) ET-1 groups. 2.0-fold increase in cardiomyocyte surface area, and 1.8-fold in protein synthesis rate in cardiomyocyte were observed after ET-1 administration and these changes were greatly prevented by estrogen pre-treatment. Estrogen could also normalize the upregulated ET-1 and ETA receptor mRNA expression in ET-1-induced hypertrophied cardiomyocyte. The pure estrogen receptor (ER) blocker, ICI182,780, failed to reverse the estrogen-mediated anti-hypertrophic effect on ET-1-induced hypertrophied cardiomyocytes suggesting the non-genomic pathway of estrogen action. Moreover, we recently found that ET-1-mediated over-expression of VEGF contributes to the development of ET-1-induced cardiomyocyte hypertrophy. Thus, subsequently the present study investigated whether VEGF system would contribute to the anti-hypertrophic action of estrogen in ET-1-induced hypertrophied cardiomyocyte. Interestingly, we found that the upregulated VEGF system in ET-1induced hypertrophied cardiomyocyte was greatly normalized by estrogen pretreatment. The present results implied that estrogen (non-genomic action) may arrest the cardiomyocyte hypertrophy through the suppression of VEGF system and demonstrate for the first time the role of estrogen and VEGF system in the prevention of ET-1-induced cardiomyocyte hypertrophy.