Rebecca I. Schneider

Oxidative stress contributes to pulmonary hypertension in the transgenic (mRen2)27 rat

The transgenic (mRen2)27 (Ren2) rat overexpresses mouse renin in extrarenal tissues, causing increased local synthesis of ANG II, oxidative stress, and hypertension. However, little is known about the role of oxidative stress induced by the tissue renin-angiotensin system (RAS) as a contributing factor in pulmonary hypertension (PH). Using male Ren2 rats, we test the hypothesis that lung tissue RAS overexpression and resultant oxidative stress contribute to PH and pulmonary vascular remodeling. Mean arterial pressure (MAP), right ventricular systolic pressure (RVSP), and wall thickness of small pulmonary arteries (PA), as well as intrapulmonary NADPH oxidase activity and subunit protein expression and reactive oxygen species (ROS), were compared in age-matched Ren2 and Sprague-Dawley (SD) rats pretreated with the SOD/catalase mimetic tempol for 21 days. In placebo-treated Ren2 rats, MAP and RVSP, as well as intrapulmonary NADPH oxidase activity and subunits (Nox2, p22phox, and Rac-1) and ROS, were elevated compared with placebo-treated SD rats (P < 0.05). Tempol decreased RVSP (P < 0.05), but not MAP, in Ren2 rats. Tempol also reduced intrapulmonary NADPH oxidase activity, Nox2, p22phox, and Rac-1 protein expression, and ROS in Ren2 rats (P < 0.05). Compared with SD rats, the cross-sectional surface area of small PA was 38% greater (P < 0.001) and luminal surface area was 54% less (P < 0.001) in Ren2 rats. Wall surface area was reduced and luminal area was increased in tempol-treated SD and Ren2 rats compared with untreated controls (P < 0.05). Collectively, the results of this investigation support a seminal role for enhanced tissue RAS/oxidative stress as factors in development of PH and pulmonary vascular remodeling.

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

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

Comparative effect of direct renin inhibition and AT1R blockade on glomerular filtration barrier injury in the transgenic Ren2 rat

Renin-angiotensin system (RAS) activation contributes to kidney injury through oxidative stress. Renin is the rate-limiting step in angiotensin (ANG II) generation. Recent work suggests renin inhibition improves proteinuria comparable to ANG type 1 receptor (AT1R) blockade (ARB). Thereby, we investigated the relative impact of treatment with a renin inhibitor vs. an ARB on renal oxidative stress and associated glomerular structural and functional changes in the transgenic Ren2 rat, which manifests hypertension, albuminuria, and increased tissue RAS activity. Young Ren2 and age-matched Sprague-Dawley (SD) controls (age 6-9 wk) were treated with a renin inhibitor (aliskiren), an ARB (irbesartan), or vehicle for 21 days. Ren2 rats exhibited increases in systolic pressure (SBP), albuminuria, and renal 3-nitrotyrosine content as well as ultrastructural podocyte foot-process effacement and diminution of the podocyte-specific protein nephrin. Structural and functional alterations were accompanied by increased renal cortical ANG II, AT1R, as well as NADPH oxidase subunit (Nox2) expression compared with SD controls. Abnormalities were attenuated to a similar extent with both aliskiren and irbesartan treatment. Despite the fact the dose of irbesartan used caused a greater reduction in SBP than aliskerin treatment (P < 0.05), the effects on proteinuria, nephrin, and oxidative stress were similar between the two treatments. Our results highlight both the importance of pressor-related reductions on podocyte integrity and albuminuria as well as RAS-mediated oxidant stress largely comparable between ARB and renin inhibition treatment.

Rosuvastatin ameliorates the development of pulmonary arterial hypertension in the transgenic (mRen2)27 rat

We have recently reported that transgenic (mRen2)27 rats (Ren2 rats) exhibit pulmonary arterial hypertension (PAH), which is, in part, mediated by oxidative stress. Since 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) exhibit beneficial vascular effects independent of cholesterol synthesis, we hypothesized that rosuvastatin (RSV) treatment ameliorates PAH and pulmonary vascular remodeling in Ren2 rats, in part, by reducing oxidative stress. Six-week-old male Ren2 and Sprague-Dawley rats received RSV (10 mg x kg(-1) x day(-)1 ip) or vehicle for 3 wk. After treatment, right ventricular systolic pressure (RVSP) and mean arterial pressure (MAP) were measured. To evaluate treatment effects on pulmonary arteriole remodeling, morphometric analyses were performed to quantitate medial thickening and cell proliferation, whereas whole lung samples were used to quantitate the levels of 3-nitrotyrosine, superoxide, stable nitric oxide (NO) metabolites [nitrates and nitrites (NO(x))], and expression of NO synthase isoforms. In the Ren2 rat, RVSP is normal at 5 wk of age, PAH develops between 5 and 7 wk of age, and the elevated pressure is maintained with little variation through 13 wk. At 8 wk of age, left ventricular function and blood gases were normal in the Ren2 rat. Ren2 rats exhibited elevations in medial hypertrophy due to smooth muscle cell proliferation, 3-nitrotyrosine, NO(x), NADPH oxidase activity, and endothelial NO synthase expression compared with Sprague-Dawley rats. RSV significantly blunted the increase in RVSP but did not reduce MAP in the Ren2 rat; additionally, RSV significantly attenuated the elevated parameters examined in the Ren2 rat. These data suggest that statins may be a clinically viable adjunct treatment of PAH through reducing peroxynitrite formation.

Sex differences in baroreflex sensitivity, heart rate variability, and end organ damage in the TGR(mRen2)27 rat

The aim of this investigation was to evaluate sex differences in baroreflex and heart rate variability (HRV) dysfunction and indexes of end-organ damage in the TG(mRen2)27 (Ren2) rat, a model of renin overexpression and tissue renin-angiotensin-aldosterone system overactivation. Blood pressure (via telemetric monitoring), blood pressure variability [BPV; SD of systolic blood pressure (SBP)], spontaneous baroreflex sensitivity, HRV [HRV Triangular Index (HRV-TI), standard deviation of the average NN interval (SDNN), low and high frequency power (LF and HF, respectively), and Poincaré plot analysis (SD1, SD2)], and cardiovascular function (pressure-volume loop analysis and proteinuria) were evaluated in male and female 10-wk-old Ren2 and Sprague Dawley rats. The severity of hypertension was greater in Ren2 males (R2-M) than in Ren2 females (R2-F). Increased BPV, suppression of baroreflex gain, decreased HRV, and associated end-organ damage manifested as cardiac dysfunction, myocardial remodeling, elevated proteinuria, and tissue oxidative stress were more pronounced in R2-M compared with R2-F. During the dark cycle, HRV-TI and SDNN were negatively correlated with SBP within R2-M and positively correlated within R2-F; within R2-M, these indexes were also negatively correlated with end-organ damage [left ventricular hypertrophy (LVH)]. Furthermore, within R2-M only, LVH was strongly correlated with indexes of HRV representing predominantly vagal (HF, SD1), but not sympathetic (LF, SD2), variability. These data demonstrated relative protection in females from autonomic dysfunction and end-organ damage associated with elevated blood pressure in the Ren2 model of hypertension.

Nebivolol improves insulin sensitivity in the TGR(Ren2)27 rat

Hypertension is often associated with increased oxidative stress and systemic insulin resistance. Use of β-adrenergic receptor blockers in hypertension is limited because of potential negative influence on insulin sensitivity and glucose homeostasis. We sought to determine the impact of nebivolol, a selective vasodilatory β₁-adrenergic blocker, on whole-body insulin sensitivity, skeletal muscle oxidative stress, insulin signaling, and glucose transport in the transgenic TG(mRen2)27 rat (Ren2). This rodent model manifests increased tissue renin angiotensin expression, excess oxidative stress, and whole-body insulin resistance. Young (age, 6-9 weeks) Ren2 and age-matched Sprague-Dawley control rats were treated with nebivolol 10 mg/(kg d) or placebo for 21 days. Basal measurements were obtained for glucose and insulin to calculate the homeostasis model assessment. In addition, insulin metabolic signaling, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, reactive oxygen species, and ultrastructural changes as evaluated by transmission electron microscopy were examined ex vivo in skeletal muscle tissue. The Ren2 rat demonstrated systemic insulin resistance as examined by the homeostasis model assessment, along with impaired insulin metabolic signaling in skeletal muscle. This was associated with increased oxidative stress and mitochondrial remodeling. Treatment with nebivolol was associated with improvement in insulin resistance and decreased NADPH oxidase activity/levels of reactive oxygen species in skeletal muscle tissue. Nebivolol treatment for 3 weeks reduces NADPH oxidase activity and improves systemic insulin resistance in concert with reduced oxidative stress in skeletal muscle in a young rodent model of hypertension, insulin resistance, and enhanced tissue RAS expression.

Pulmonary Hemodynamic Response to Acute Combination and Monotherapy With Sildenafil and Brain Natriuretic Peptide in Rats With Monocrotaline-Induced Pulmonary Hypertension

Background:The lungs of patients with pulmonary arterial hypertension (PAH) exhibit decreased bioavailability of nitric oxide and downstream signaling through cyclic guanosine monophosphate (cGMP). Therapies that enhance cGMP-mediated vasodilation have shown efficacy in treating PAH. We tested the hypothesis that combination therapy with sildenafil, a cGMP phosphodiesterase type 5 inhibitor, and brain natriuretic peptide (BNP), a receptor-mediated guanosine cyclase stimulator, synergistically attenuates monocrotaline-induced PAH in rats compared with either monotherapy. Methods:Adult male Sprague-Dawley rats were subcutaneously injected with monocrotaline (n = 41, 50 mg/kg). After approximately 4 weeks, the rats were infused intravenously with vehicle solution, sildenafil (42 and 85 &mgr;g/kg/min), or BNP (50 and 100 ng/kg/min), alone and in varied combination. The primary endpoint was the relative change in right ventricular systolic pressure (RVSP) and mean arterial systemic pressure (MAP). Secondary endpoints included heart rate and dP/dt. Results:Vehicle infusions did not alter hemodynamic variables. Sildenafil85 (85 &mgr;g/kg/min) alone decreased RVSP (−16.6 ± 5.6%) and decreased MAP (−4.0 ± 4.7%). BNP50 (50 ng/kg/min) and BNP100 (100 ng/kg/min) decreased RVSP (−23.3 ± 5.7% and −27.1 ± 2.9%, respectively) and MAP (−6.4 ± 5.8% and −14.3 ± 4.1%, respectively). Combination therapy with sildenafil42 and BNP50 decreased RVSP (−20.7 ± 5.6%) and showed a lessened systemic effect (MAP = −11.6 ± 5.9%). Combination therapy with sildenafil85 and BNP100 decreased RVSP (−27.6 ± 3.2%, P = NS) and showed increased systemic effect (MAP = −20.7 ± 3.1%, P < 0.05) in comparison with sildenafil85. Conclusions:This study suggests that intravenous administration of both sildenafil and BNP monotherapy produces significant improvement in RVSP, making them potentially viable options for the treatment of PAH, whereas combination therapy produces no additional improvement in pulmonary hemodynamics.