High salt intake contributes to the development of hypertension in salt-sensitive humans and animals and the mechanistic causes are poorly understood. In Dahl salt-sensitive (S) but not salt-resistant (R) rats, high salt diet increases cerebrospinal fluid (CSF) [Na+] and activates an aldosterone-mineralocorticoid receptor-epithelial sodium channel-endogenous ouabain (MR-ENaC-EO) neuromodulatory pathway in the brain that enhances the activity of sympatho-excitatory angiotensinergic and glutamatergic pathways, leading to an increase in sympathetic nerve activity (SNA) and blood pressure (BP). We hypothesize that high salt diet in Dahl S rats enhances Ang II release in the paraventricular nucleus (PVN), causing a decrease in local nitric oxide (NO) action and an increase in local glutamate release thereby elevating SNA, BP and heart rate (HR). The present study evaluated the effects of agonists or blockers of MR, ENaC, EO, nitric oxide synthase (NOS) or glutamate and AT1-receptors on the BP and HR responses to acute infusions of Na+ rich aCSF, intracerebroventricularly (icv), or in the PVN of Dahl S, R or Wistar rats or to high salt diet in Dahl S and R rats. In Wistar rats, aldosterone in the PVN enhanced the BP and HR responses to infusion of Na+ rich aCSF in the PVN, but not in the CSF, and only the enhancement was prevented by blockers of MR, ENaC and EO in the PVN. AT1-receptor blockers in the PVN fully blocked the enhancement by aldosterone and the responses to infusion of Na+ rich aCSF icv, or in the PVN. Na+ rich aCSF in the PVN caused larger increases in BP and HR in Dahl S vs. R rats and the responses to Na+ were fully blocked by an AT1-receptor blocker in the PVN. BP and HR responses to a NOS blocker in the PVN were the same, but L-NAME enhanced Na+ effects more in Dahl R than S rats. High salt diet attenuated increases in BP from L-NAME in the PVN of Dahl S but not R rats. AT1 and glutamate receptor blockers candesartan and kynurenate in the PVN decreased BP in Dahl S but not R rats on high salt diet. At the peak BP response to candesartan, kynurenate in the PVN further decreased BP whereas candesartan did not further decrease BP at the peak BP response to kynurenate. Our findings indicate that both an acute increase in CSF [Na+] and high salt intake in Dahl S rats increases AT1-receptor activation and decreases NO action in the PVN thereby contributing to the pressor responses to Na+ and presumably, to dietary salt-induced hypertension. The increased BP response to AT1-receptor activation in the PVN of Dahl S is mediated by enhanced local glutamate receptor activation. An MR-ENaC-EO pathway in the PVN can be functionally active and further studies need to assess its role in Dahl S rats on high salt intake.

The incidence of chronic kidney disease (CKD) is increasing. CKD is characterized by a gradual decrease in renal function leading to end stage renal disease (ESRD). Damage to the glomerular podocytes, is one of the first hallmarks of CKD. We hypothesized that podocyte prostaglandin E2 (PGE2) receptors contribute to the progression of glomerular injury in models of CKD. To test this hypothesis, transgenic mice were generated with either podocyte-specific overexpression or deletion of the PGE2 EP4 receptor (EP4pod+and EP4pod-/- respectively). Mice were next tested in the 5/6 nephrectomy (5/6 Nx) or angiotensin II (Ang II) models of CKD. These studies revealed increased proteinuria and decreased survival for EP4pod+ mice while EP4pod-/- mice were protected against the development of glomerular injury. Furthermore, our findings were supported by in vitro studies using cultured mouse podocytes where an adhesion defect was uncovered for cells overexpressing the EP4 receptor. Additionally, our investigations have demonstrated a novel synergy between angiotensin II AT1 receptors and prostaglandin E2 EP4 receptors. This was revealed by in vitro studies using isolated mouse glomeruli. There we were able to show that Ang II stimulation leads to increased expression of cyclooxygenase 2 (COX-2), the enzyme responsible for synthesis of PGE2, in a p38 mitogen activated protein kinase (MAPK) dependent fashion. Moreover increased PGE2 synthesis was measured in response to Ang II stimulation. We confirmed the presence of this synergy in our cultured mouse podocytes and showed an adhesion defect in response to Ang II stimulation which was COX-2 and EP4 dependent. These findings suggest that Ang II AT1 receptors and PGE2 EP4 receptors act in concert to exacerbate glomerulopathies. Studies using mice with either podocyte-specific overexpression of a dominant negative p38 MAPK or mice with global deletion of the EP1 receptor did not provide conclusive results as to their respective signaling involvement in podocyte injury. Altogether our findings provide novel insight for podocyte PGE2 EP4 and Ang II AT1 receptor signaling in models of CKD. These studies provide novel avenues for pursuing therapeutic interventions for individuals with progressive kidney disease.

The Angiotensin II Type 1 (AT1) receptor is the main receptor responsible for the effects of the renin-angiotensin system, and its expression pattern is altered in several diseases. [11C]Methyl-Losartan has been developed based on the clinically used AT1 receptor antagonist Losartan. The aim of this work is to characterize the pharmacokinetics, repeatability and reliability of measurements, binding specificity and selectivity of [11C]Methyl-Losartan in rats using in vivo small animal positron emission tomography (PET) imaging, ex vivo biodistribution and in vitro autoradiography methods. Also, we aim to measure the presence of metabolites in the kidney and plasma using high-performance liquid chromatography. We have demonstrated in vivo that [11C]Methyl-Losartan is taken up in the AT1 receptor-rich kidneys and that it is displaceable by selective AT1 receptor antagonists. Using ex vivo biodistribution, we have confirmed these results and demonstrated that [11C]Methyl-Losartan binds selectively to the AT1 receptor over the AT2, Mas and β-adrenergic receptors. In vitro autoradiography results confirmed these renal binding selectivity studies. [11C]Methyl-Losartan was also shown to have one and two C-11 labeled metabolites in the plasma and kidneys, respectively. In conclusion, [11C]Methyl-Losartan is a promising agent for studying the AT1 receptor in rat models with normal and altered AT1 receptor expression using small animal PET imaging.

This study was designed to test the hypothesis that 60 days continuous head down bed rest (HDBR), an Earth-based analogue of the effects of space flight, would elevate factors that increase vasoconstriction and would increase markers of vascular inflammation. The study incorporated countermeasures consisting of treadmill running within lower-body negative pressure and resistive "flywheel" exercise (exercise countermeasure, EX) or an increased protein intake of 0. 6 g/kg body weight/day (dietary countermeasures, DIET) to determine whether these interventions might prevent the vasoconstrictor and inflammatory responses when compared to a control (CON) group. Markers of vascular health measured in the study include the vasoactive molecules angiotensin II (Ang II), endothelin-1 (ET-1), and nitric oxide metabolites (NO_met); and markers of inflammation including C-reactive protein (CRP), and the adhesion molecules E-selectin (E-sel), intracellular adhesion molecule-1 (ICAM), and vascular cell adhesion molecule-1 (VCAM). Twenty four women were housed at the MEDES clinic in Toulouse, France, as part of a large international study (Women International Space Simulation for Exploration, WISE) in which various experimental protocols and countermeasures were integrated into a single experimental design completed during two campaigns. Each 100 day campaign included 20 days of pre-testing (pre-HDBR), 60 days of bed rest (HDBR), and 20 days of post-testing (post-HDBR). The experimental countermeasures were applied only during the 60-day HDBR period. Following 60 days of HDBR, many changes occurred in the concentrations of the measured molecules. Specifically, the concentration of Ang II significantly increased in the CON and DIET groups (52. 9%, p = 0. 014; and 124. 4%, p <0. 0001 respectively), but not in the EX group. Also, NO_met decreased in all groups, with reductions in the EX and DIET groups (p = 0. 013, and p = 0. 056 respectively). Markers used to assess vascular inflammation increased following the HDBR. The increase in CRP in the CON and DIET groups and the decrease in the EX group from pre- to post-HDBR were not significant; however, the directional changes resulted in an interaction between group and HDBR (p = 0. 052). The adhesion molecule E-sel was significantly increased in the DIET group (p = 0. 003), and VCAM was significantly increased in the CON group (p = 0. 016) with a smaller increase in the DIET group (p = 0. 08). No changes in adhesion molecules were observed in the EX group. This study demonstrated that 60 days of HDBR by young, healthy, women caused changes in several different molecules that are beginning to emerge as risk factors for the development of cardiovascular diseases. Further, it was observed that regular, vigorous exercise during HDBR prevented these changes. These results suggest that future studies of this kind should directly monitor the effects of simulated space flight on vascular health in men and women to obtain a greater understanding of the adaptations that might occur during long term space exploration missions. HDBR can be considered an extreme model of physical inactivity and could be used to provide insight into mechanisms of disease processes associated with the sedentary lifestyle that is prevalent in Western society.

In this study, we investigated the role of Ang II in pathological vascular remodeling. We sought to determine whether the humoral or the mechanical effects of Ang II are the dominant factor driving the remodeling process. The following experimental groups were used: control group (untreated mice), mice treated with an angiotensin receptor blocker (Candesartan, 0.5 mg/kg/day,SQ), an ACE inhibitor (Captopril, 6 mg/kg/day), and a calcium-channel blocker (Amlodipine, 7.5 mg/kg/day). All mice (n=6 per experimental group) were from the C57Bl/6 background. The carotid ligation model was implemented to study the differences in vascular remodeling. Additionally, multiple time points (7-, 14-, and 21-days post-surgery) were used to track the progression of remodeling. In Day-7 analysis, all three treatment groups yielded similar remodeling patterns as evidenced by a significant reduction in neointimal area, medial thickening and hypertrophy compared with the control group. Histomorphometric analysis of carotid sections collected 1mm below the ligation demonstrated that the Amlodipine group had 26% reduction in total vessel area, Candesartan a 36% reduction, and Captopril a 28% reduction (p less than 0.05 in all groups compared with Control), as well as a parallel 38-40% drop in medial thickness. In Day-14 analysis, no significant differences between the Controls and treatment groups were observed, although differences were emerging between the treatment groups. Candesartan was found to reduce the extent of negative remodeling observed between the 7- and 14-day Control data, whereas the Captopril group did not exhibit this trend. All treatment groups exhibited less neointimal formation than Controls, similar to Day-7. By the 21-day time point, the Captopril group underwent positive remodeling, resembling the Candesartan and Amlodipine groups. Although total vessel area was analogous among all groups, neointimal areas were significantly decreased in the treatment groups. Blood pressure plays a pivotal role in the modulation of vascular remodeling in response to mechanical injury. Although intermediate timepoint analysis suggests that humoral aspects of ACE inhibition or angiotensin-receptor blockade yielded unique effects on the overall vessel caliber, upon reaching the late, 21-day time point, the mechanical factors became predominant. These data support the importance of blood pressure control in the attenuation of pathological vascular remodeling.

Angiotensin II (Ang II) exerts diverse physiological actions in both peripheral and central nervous system. It has been demonstrated to implicate in central mechanisms leading to hypertension in the nucleus tractus solitarii (NTS) of rats, and mediated by the type-1 receptors (AT1R). Our previous studies already suggested that inhibition of NO synthesis in the NTS causes sustained hypertension. It was reported that the activity of Ang II was higher in the NTS of spontaneously hypertensive rat (SHR) and AT1R are colocalized in the neurons of the NTS, providing the local reactive oxygen species (ROS) production by Ang II. However, the signaling mechanisms of Ang II that induce hypertension remain uncertain. In the present study, we investigated the possible signal pathways involved in the cardiovascular regulation of Ang II in the NTS. Male SHR was treated with AT1R blocker, losartan (30 mg/kg/day) or superoxide dismutase (SOD) mimetic, tempol (1 mM/kg/day) for two weeks, systolic blood pressure was decreased significantly in losartan- or tempol-treated SHR. The NTS was excised for dihydroethidium (DHE) staining, NO analysis, immunoblotting and immunohistochemistry. Our results demonstrated that DHE staining revealed of ROS was much more in the NTS of SHR than in the NTS of wistar-Kyoto (WKY) rat. The ROS in the NTS of SHR was reduced by losartan. The NO content in the NTS of SHR was lower than WKY, while losartan and tempol could increase NO in the NTS of SHR. Immunoblotting and immunohistochemistry studies demonstrated that Ang II-induced hypertension inhibited neuronal NO synthase (nNOS), ERK and RSK phosphorylation levels in the NTS of SHR. These results suggest that Ang II induces ROS production in the NTS of SHR. In addition, the cardiovascular modulatory effects of Ang II in the NTS are accomplished by downregulation of ERK1/2-RSK phosphorylation levels and then nNOS level.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

A better understanding of these structural changes that occur before Hypertension (HTN) could ultimately result in a treatment that can prevent or reverse this disease state before its onset. T cells have been shown essential for the development of HTN. The aim of these murine studies was to investigate the role of the T-helper CD4⁺ lymphocytes in initiating vascular remodeling and HTN in the absence of an increased mechanical load and to investigate the role of T-helper 17 (Th17) CD4⁺ lymphocyte initiating vascular remodeling and HTN by stimulating Lysyl Oxidase (LOX). LOX is known to cross-link collagen and elastin and. Excess synthesis of collagen and elastin results in a stiffer artery and hypertension. We established L-NAME-induce HTN in wild type (WT) mice. CD4⁺ splenic lymphocytes were isolated from these mice and adoptively transferred into naïve syngeneic severe combined immunodeficient (SCID) mice. The SCID mice receiving these cells became hypertensive. Cytokine analysis demonstrated an increase in both Th1 and Th17 cytokine in HTN donor mice and of lymphocytes in the aortic infiltrates of the HTN recipient mice. The increased collagen and LOX expression in recipient mice suggest that the adoptively transfer CD4⁺ lymphocytes are associated with vascular extracellular matrix remodeling. Furthermore we examined the role of Th17 lymphocyte in aortic LOX regulation in Angiotensin II-induced hypertension. The Increase in blood pressure and Velocity Time Integral (VTI) was measured in WT Angiotensin II treated mice whereas no change was detected in the Th17 deficient (RORT KO) Angiotensin II treated group. When compared to the control group the WT group infused with Angiotensin II had higher LOX protein expression, LOX fluorescent Immunohistochemical stain and LOX activity. This group also had increased hydroxyproline levels, collagen stain, hyperplasia and aortic thickening. In contrast, the Th17 deficient mice Angiotensin II treated group had no changes in these parameters. The results provide evidence that IL-17 mediates Angiotensin II-induced hypertension and vascular dysfunction by the overstimulation of LOX. Potentially targeting T17 cells will allow for a drug-specific therapeutic approach and delay the progression of hypertension pathology.

Renal arterioles represent the most important effecter site in the control of renal perfusion and filtration. Adenosine (Ado), angiotensin II (Ang II) and nitric oxide (NO) interact in modulating arteriolar tone. The present work investigates the mechanism of this interaction. We tested the hypothesis that AT1 receptor (AT1AR) mediated NO release in isolated perfused afferent arterioles. Further, special attention was given to mechanisms of Ado-Ang II -interactions. We found (I) that Ang II specifically induces NO release via AT1AR in arterioles. The effect is important in view of high renin and Ang II concentrations in these vessels. (II) Ado modulates the Ang II response by acting on vasoconstrictor A1AR and vasodilator A2AR. Vice versa, Ang II critically enhances the constriction to Ado, which supports the assumption of its modulating action in the tubuloglomerular feedback (TGF). (III) The synergistic effect of Ang II and Ado on arteriolar contraction is concurrent with an increase in the cytosolic calcium. Further, (IV) Ado increases the calcium sensitivity of the contractile machinery in arteriolar smooth muscle cells most probably by enhancement of the phosphorylation of the myosin light chain regulatory unit. RhoA kinase, protein kinase C and p38 MAP are involved in the Ado effect, which is not receptor mediated and depends on the Ado uptake into vascular cells. Remarkably, the enhancing action of Ado is most likely limited to Ang II; since Ado does not influence endothelin-1 and norepinephrine induced contractions. These novel results extend our knowledge about the synergistic action of Ang II and Ado in the control of renal filtration. Ado, the key factor in mediation of the TGF, develops a significant vasoconstrictor action only in the presence of Ang II. On the other hand, the Ang II induced vasoconstriction is modulated by Ado via receptor and non-receptor mediated intracellular signaling pathways.

No description available.