Chronic Nitric Oxide Synthase Research Articles

#4544 THE ASSOCIATION OF TAMOXIFEN TO THE CONSERVATIVE CKD TREATMENT PROMOTED ADDITIONAL ANTIFIBROTIC EFFECTS IN A MODEL OF HYPERTENSIVE NEPHROSCLEROSIS

Abstract Background and Aims Chronic kidney disease (CKD) is an insidious, progressive and highly debilitating condition, which leads to the loss of renal function and to the need of life-sustaining renal replacement therapy. The conservative treatment of CKD is mainly based in the blockade of the renin-angiotensin-aldosterone system (RAAS), which can be associated to immunosuppressant drugs, according to the etiology of the renal injury. However, this pharmacological approach is not able to prevent CKD progression completely. Until the present moment, medical community still lack of a specific drug to effectively stop the progression of renal fibrosis associated to CKD, which once triggered became irreversible. Tamoxifen (TAM) is an estrogen receptor antagonist, widely employed for the clinical treatment of breast cancer, and responsible for saving a number of lives worldwide, in the past decades. This well-tolerated and cost-effective drug have been reported to exert antifibrotic effects in both experimental and human peritoneal fibrosis. Moreover, our research group already demonstrated that TAM efficiently prevented albuminuria, glomerulosclerosis, and interstitial fibrosis in a model of CKD. In the present study, we investigated if the association of TAM to the classic conservative treatment of CKD, here obtained by the association of Losartan (LOS) and Micofenolate Mofetil (MMF), could promote further renoprotection in an experimental model of hypertensive nephrosclerosis, induced by the chronic nitric oxide synthase blockade, obtained by the oral administration of L-NAME (NAME), associated to a high sodium (HS) diet, in rats. Method The experimental protocol was approved by the local Research Ethics Committee (CAPPesq) and was developed in strict conformity with the international standards for care and manipulation of laboratory animals. Thirty male Wistar rats were kept with a 3.2% HS diet for 15 days of adaptation, before the protocol start. After this period, the animals continued to receive the HS diet and were divided among 5 groups. CONT: receiving no further drugs or treatments, NAME: treated with 70 mg/kg/d of L-NAME, diluted in drinking water, LOS: NAME rats treated with 50mg/Kg/d of LOS, also diluted in drinking water, MMF: NAME rats treated with 10 mg/Kg/d of MMF given by gavage, TAM: NAME rats treated with 10 mg/Kg/d also by gavage, and LOS+MMF+TAM: NAME rats treated with all the drugs simultaneously. Systolic blood pressure (SBP), urinary albumin excretion (24 h uAE), glomerulosclerosis (GS), glomerular ischemia (GI), interstitial fibrosis (INT), tubulointerstitial infiltration of macrophages (CD68) and T-cells (CD3), as well as renal cortical interstitial collagen I (COLL1) and fibronectin (FIBRO) accumulation were evaluated after 30 days of treatment. Results The association of TAM to the classic treatment of CKD improved the renoprotection obtained by LOS+MMF. The triple combined treatment significantly reduced hypertension, albuminuria, glomerular structural damage, renal macrophage and T-cell infiltration in NAME rats, compared to the animals treated with the respective monotherapies. Moreover, both TAM alone or the association, were equally effective in reducing interstitial collagen and fibronectin accumulation, in NAME rats. Data are presented as Mean ± SE. For One-way ANOVA statistical analysis, we considered: p<0.05 vs.*CONT, #NAME, †LOS, §MMF, &TAM. Conclusion Our preliminary results shown TAM to be effective in reducing renal inflammation and fibrosis in the chronic nitric oxide synthase blockade model and to exert additional renoprotective effects when associated to LOS and MMF in all the analyzed parameters. Although further studies with different nephropathy models are still required in order to confirm our findings, here we suggest TAM to be a potential adjuvant in the conservative management of CKD.

Suppression of nitric oxide synthase aggravates non-alcoholic steatohepatitis and atherosclerosis in SHRSP5/Dmcr rat via acceleration of abnormal lipid metabolism.

Non-alcoholic steatohepatitis (NASH) is a progressive subtype ofnon-alcoholic fatty liver disease (NAFLD) that is closely related to cardiovascular disease (CVD). Nitric oxide (NO) plays a critical role in the control of various biological processes. Dysfunction of the NO signaling pathway is associated with various diseases such as atherosclerosis, vascular inflammatory disease, and diabetes. Recently, it has been reported that NO is related to lipid and cholesterol metabolism. Chronic NO synthase (NOS) inhibition accelerates NAFLD by increasing hepatic lipid deposition. However, the detailed relationship between NO and abnormal lipid and cholesterol metabolism in NAFLD/NASH has not been completely explained. We aimed to determine the effects of NOS inhibition by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME), a NOS inhibitor, on NASH and CVD via lipid and cholesterol metabolism. Stroke-prone spontaneously hypertensive rats were fed a high-fat and high-cholesterol diet for 8weeks and administered L-NAME for the last 2weeks. Following blood and tissue sampling, biochemical analysis, histopathological staining, quantitative RT-PCR analysis, and western blotting were performed. L-NAME markedly increased hepatic triglyceride (TG) and cholesterol levels by promoting TG synthesis and cholesterol absorption from the diet. L-NAME increased the mRNA levels of inflammatory markers and fibrotic areas in the liver. Cholesterol secretion from the liver was promoted in rats administered L-NAME, which increased serum cholesterol. L-NAME significantly increased the level of oxidative stress marker and lipid deposition in the arteries. NOS inhibition simultaneously aggravates NASH and atherosclerosis via hepatic lipid and cholesterol metabolism.

Protective role of the mitochondrial fusion protein OPA1 in hypertension.

Hypertension is associated with excessive reactive oxygen species (ROS) production in vascular cells. Mitochondria undergo fusion and fission, a process playing a role in mitochondrial function. OPA1 is essential for mitochondrial fusion. Loss of OPA1 is associated with ROS production and cell dysfunction. We hypothesized that mitochondria fusion could reduce oxidative stress that defect in fusion would exacerbate hypertension. Using (a) Opa1 haploinsufficiency in isolated resistance arteries from Opa1+/- mice, (b) primary vascular cells from Opa1+/- mice, and (c) RNA interference experiments with siRNA against Opa1 in vascular cells, we investigated the role of mitochondria fusion in hypertension. In hypertension, Opa1 haploinsufficiency induced altered mitochondrial cristae structure both in vascular smooth muscle and endothelial cells but did not modify protein level of long and short forms of OPA1. In addition, we demonstrated an increase of mitochondrial ROS production, associated with a decrease of superoxide dismutase 1 protein expression. We also observed an increase of apoptosis in vascular cells and a decreased VSMCs proliferation. Blood pressure, vascular contractility, as well as endothelium-dependent and -independent relaxation were similar in Opa1+/- , WT, L-NAME-treated Opa1+/- and WT mice. Nevertheless, chronic NO-synthase inhibition with L-NAME induced a greater hypertension in Opa1+/- than in WT mice without compensatory arterial wall hypertrophy. This was associated with a stronger reduction in endothelium-dependent relaxation due to excessive ROS production. Our results highlight the protective role of mitochondria fusion in the vasculature during hypertension by limiting mitochondria ROS production.

The investigation of antioxidant and anti-inflammatory potentials of apitherapeutic agents on heart tissues in nitric oxide synthase inhibited rats via Nω-nitro-L-arginine methyl ester

ABSTRACT Background High blood pressure effects heart and vessels. Development of pathogenesis is the result of oxidative stress. We aimed to investigate the antioxidant effects of propolis, caffeic acid phenethyl ester (CAPE), and pollen on the hearts of rats which chronic nitric oxide synthase (NOS) inhibited through Nω-nitro-L-arginine methyl ester (L-NAME). Paraoxonase 1 (PON1), total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), asymmetric dimethylarginine (ADMA), and nuclear factor-κB (NF-κB) were analyzed on the heart. Material and Methods Sprague-Dawley rats were divided five groups of seven rats in every group; Group I: Control, Group II: L-NAME, Group III: L-NAME+propolis, Group IV: L-NAME+CAPE and Group V: L-NAME+pollen. L-NAME become dissolved in regular saline (0.9% NaCl w/v). The ethanolic extract of propolis (200 mg/kg/days, gavage), pollen (100 mg/kg/days, by gavage), CAPE (50 µM/kg/days, intraperitoneally), and the NOS inhibitor L-NAME (40 mg/kg, intraperitoneally) had been administered. Results Blood pressure (BP) of rats treated with propolis, CAP,E and pollen statistically significant decreased. Decreasing in BP of the rats of pollen group was more than CAPE and propolis groups (P < .05). PON1 and TAS levels decreased in L-NAME-treated groups (P < .05), but ranges have been better in propolis, CAPE and pollen groups. TOS, ADMA and NF-κB levels increased (P < .05) in L-NAME group; however, these parameters were lower (P < .05) in propolis and CAPE groups (P < .05). Conclusions Vasorelaxant properties and free radical scavenging actions of propolis, CAPE, and pollen may reduce the oxidative stress and blood pressure in the rats chronic NOS inhibited through L-NAME.

Cardiac remodelling in a swine model of chronic thromboembolic pulmonary hypertension: comparison of right vs. left ventricle.

Key points Right ventricle (RV) function is the most important determinant of survival and quality of life in patients with chronic thromboembolic pulmonary hypertension (CTEPH).The changes in right and left ventricle gene expression that contribute to ventricular remodelling are incompletely investigated.RV remodelling in our CTEPH swine model is associated with increased expression of the genes involved in inflammation (TGFβ), oxidative stress (ROCK2, NOX1 and NOX4), and apoptosis (BCL2 and caspase‐3).Alterations in ROCK2 expression correlated inversely with RV contractile reserve during exercise.Since ROCK2 has been shown to be involved in hypertrophy, oxidative stress, fibrosis and endothelial dysfunction, ROCK2 inhibition may present a viable therapeutic target in CTEPH. Right ventricle (RV) function is the most important determinant of survival and quality of life in patients with chronic thromboembolic pulmonary hypertension (CTEPH). The present study investigated whether the increased cardiac afterload is associated with (i) cardiac remodelling and hypertrophic signalling; (ii) changes in angiogenic factors and capillary density; and (iii) inflammatory changes associated with oxidative stress and interstitial fibrosis. CTEPH was induced in eight chronically instrumented swine by chronic nitric oxide synthase inhibition and up to five weekly pulmonary embolizations. Nine healthy swine served as a control. After 9 weeks, RV function was assessed by single beat analysis of RV–pulmonary artery (PA) coupling at rest and during exercise, as well as by cardiac magnetic resonance imaging. Subsequently, the heart was excised and RV and left ventricle (LV) tissues were processed for molecular and histological analyses. Swine with CTEPH exhibited significant RV hypertrophy in response to the elevated PA pressure. RV–PA coupling was significantly reduced, correlated inversely with pulmonary vascular resistance and did not increase during exercise in CTEPH swine. Expression of genes associated with hypertrophy (BNP), inflammation (TGFβ), oxidative stress (ROCK2, NOX1 and NOX4), apoptosis (BCL2 and caspase‐3) and angiogenesis (VEGFA) were increased in the RV of CTEPH swine and correlated inversely with RV–PA coupling during exercise. In the LV, only significant changes in ROCK2 gene‐expression occurred. In conclusion, RV remodelling in our CTEPH swine model is associated with increased expression of genes involved in inflammation and oxidative stress, suggesting that these processes contribute to RV remodelling and dysfunction in CTEPH and hence represent potential therapeutic targets.

Chronic inhibition of nitric oxide synthase modulates calcium handling in rat heart 1.

Systemic infusion of nitric oxide synthase (NOS) inhibitors increases peripheral vascular resistance due to inhibition of endothelial NOS leading to the activation of the arterial baroreceptor mechanisms and inhibition of central sympathetic outflow. In the current study, we explored that systemic NOS blockage activates protein kinase A (PKA)-mediated signaling pathway through maintained cGMP-dependent protein kinase (PKG) activation. Rats were treated with 3 different concentrations of N(ω)-nitro-l-arginine methyl ester (L-NAME) for 14 days. Systemic L-NAME treatment induced a dose-dependent increase in blood pressure and increased mRNA levels of atrial natriuretic peptide (ANP) and phosphorylation levels of p44/42 MAPK without any change in cardiac mass. The cardiac cGMP levels and PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) (Ser239) did not alter in any group. At the highest dose of treatment (100 mg/kg per day), PKA-mediated phosphorylations of VASP (Ser157) and troponin I (TnI) (Ser23/24) were enhanced significantly indicating the increase in PKA activation in response to chronic NOS blockage. Alterations in both phosphorylated phospholamban (Ser16/Thr17) and sarcoplasmic/endoplasmic Ca2+-ATPase (SERCA2) levels can increase cytosolic Ca2+ load and impair Ca2+ handling. Our data suggest that the increased PKA activation in response to chronic NOS blockage appears to be responsible for cardiac abnormalities that occur due to prolonged L-NAME treatment.

The Probiotic Lactobacillus fermentum Prevents Dysbiosis and Vascular Oxidative Stress in Rats with Hypertension Induced by Chronic Nitric Oxide Blockade

Whether the probiotic Lactobacillus fermentum CECT5716 (LC40) ameliorates hypertension in rats with chronic nitric oxide (NO) synthase inhibition is tested. Rats are randomly divided into four different groups and treated for 4weeks: a) vehicle (control), b) vehicle plus NG -nitro-l-arginine methyl ester (l-NAME; 50mg100mL-1 in drinking water), c) LC40 (109 colony-forming unitsd-1 by gavage), and d) LC40 plus l-NAME. l-NAME induces gut dysbiosis, characterized mainly by an increased Fimicutes/Bacteroidetes (F/B) ratio and reduced Bifidobacterium content, increased Th17 cells and reduced Treg in mesenteric lymph nodes (MLN), increased aortic Th17 infiltration and reactive oxygen species, reduced aortic endothelium-dependent relaxant response to acetylcholine, and hypertension. LC40 prevents gut dysbiosis, alters the Th17/Treg balance in MLN, vascular oxidative stress, and inflammation, slightly improves endothelial dysfuncion but do not inhibit the development of l-NAME-induced hypertension. Chronic LC40 treatment, in this model of chronic inhibition of NO synthesis, reduces early events involved in atherosclerosis development, such as vascular oxidative stress and pro-inflammatory status, as a result of prevention of gut dysbiosis and immune changes in MLN, but not hypertension, confirming the critical role of NO in the antihypertensive effects of LC40 in genetic hypertension.

Nitric Oxide Synthase Inhibition Induces Renal Medullary Hypoxia in Conscious Rats.

BackgroundRenal hypoxia, implicated as crucial factor in onset and progression of chronic kidney disease, may be attributed to reduced nitric oxide because nitric oxide dilates vasculature and inhibits mitochondrial oxygen consumption. We hypothesized that chronic nitric oxide synthase inhibition would induce renal hypoxia.Methods and ResultsOxygen‐sensitive electrodes, attached to telemeters, were implanted in either renal cortex (n=6) or medulla (n=7) in rats. After recovery and stabilization, baseline oxygenation (pO 2) was recorded for 1 week. To inhibit nitric oxide synthase, N‐ω‐nitro‐l‐arginine (L‐NNA; 40 mg/kg/day) was administered via drinking water for 2 weeks. A separate group (n=8), instrumented with blood pressure telemeters, followed the same protocol. L‐NNA rapidly induced hypertension (165±6 versus 108±3 mm Hg; P<0.001) and proteinuria (79±12 versus 17±2 mg/day; P<0.001). Cortical pO 2, after initially dipping, returned to baseline and then increased. Medullary pO 2 decreased progressively (up to −19±6% versus baseline; P<0.05). After 14 days of L‐NNA, amplitude of diurnal medullary pO 2 was decreased (3.7 [2.2–5.3] versus 7.9 [7.5–8.4]; P<0.01), whereas amplitudes of blood pressure and cortical pO 2 were unaltered. Terminal glomerular filtration rate (1374±74 versus 2098±122 μL/min), renal blood flow (5014±336 versus 9966±905 μL/min), and sodium reabsorption efficiency (13.0±0.8 versus 22.8±1.7 μmol/μmol) decreased (all P<0.001).ConclusionsFor the first time, we show temporal development of renal cortical and medullary oxygenation during chronic nitric oxide synthase inhibition in unrestrained conscious rats. Whereas cortical pO 2 shows transient changes, medullary pO 2 decreased progressively. Chronic L‐NNA leads to decreased renal perfusion and sodium reabsorption efficiency, resulting in progressive medullary hypoxia, suggesting that juxtamedullary nephrons are potentially vulnerable to prolonged nitric oxide depletion.

The Effect of Chronic NO Synthase Inhibition on the Vasoactive and Structural Properties of Thoracic Aorta, NO Synthase Activity, and Oxidative Stress Biomarkers in Young SHR.

Although the role of nitric oxide (NO) in essential hypertension is still unclear, the effects of long-term NO deficiency have not yet been investigated during the critical juvenile period in spontaneously hypertensive rats (SHR). We aimed to analyze the effects of chronic NO synthase (NOS) inhibition on systolic blood pressure (sBP), vasoactivity, morphological changes and superoxide level in the thoracic aorta (TA), NOS activity in different tissues, and general biomarkers of oxidative stress in plasma of young SHR. Four-week-old SHR were treated with NG-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg/day, p.o.) for 4-5 weeks. L-NAME treatment induced a transient sBP increase only, and surprisingly, slightly inhibited endothelium-dependent relaxation of TA. Hereby, the inhibition of NOS activity varied from tissue to tissue, ranging from the lowest in the TA and the kidney to the highest in the brain stem. In spite of an increased sensitivity of adrenergic receptors, the maximal adrenergic contraction of TA was unchanged, which was associated with changes in elastin arrangement and an increase in wall thickness. The production of reactive oxygen species in the TA was increased; however, the level of selected biomarkers of oxidative stress did not change. Our findings proved that the TA of young SHR responded to chronic NO deficiency by the development of adaptive mechanisms on the functional (preserved NO-derived vasorelaxation, unincreased contraction) and molecular (preserved NOS activity) level.

Early life stress (ELS) protects against LNAME hypertension‐induced renal tubular damage

In humans, early life stress (ELS) is a newly recognized cardiovascular disease risk factor and associated with increases in inflammation and risk of hypertension. Utilizing a rat model of ELS, maternal separation (MatSep), we recently reported that MatSep induced immune activation in the kidney compared to normally‐reared control animals. Chronic nitric oxide synthase blockade with LNAME in the drinking water is a model of hypertension with increased immune cells and inflammation. Therefore, we hypothesized that exposure to MatSep during early life and LNAME during adulthood would induce increased levels of renal injury markers and immune cells when compared to normally‐reared rats with LNAME during adulthood. MatSep involves separating rat pups from the dam for 3hrs/day from postnatal day 2 (PD2) to PD14. Normally‐reared littermate rats were used as controls. Twelve weeks old adult (n=6–8rats/group) control and MatSep rats were given LNAME (100mg/kg/rat) in drinking water for 7 days. LNAME was adjusted daily based on the amount of water intake. We compared urinary renal damage markers and renal immune cell populations in control and MatSep rats after LNAME treatment. On day 7 after LNAME, urine was collected for assessment of glomerular (albumin and nephrin) and tubular (KIM1 and NGAL) injury markers by enzyme‐linked immunosorbent assay (ELISA). Our results showed that urinary KIM1 excretion was significantly lower in MatSep rats compared to control rats (control LNAME vs. MatSep LNAME: 6.78mg/day ± 0.32 vs. 5.53mg/day ± 0.36; p = 0.029). No statistical differences were observed in urinary NGAL, albumin and nephrin excretion. At day 7 after LNAME, blood and kidneys were used for determination of immune cell populations by flow cytometry. The proportion of renal total T cells, T helper cells, macrophages, neutrophils, natural killer, and dendritic cells were similar in control and MatSep rats. However, there was a trend towards an increase in the proportion of cytotoxic T cells in MatSep compared to control animals (%CD45+ cells; control LAME vs. MatSep LNAME: 14.04 ± 0.97 vs. 17.37 ± 1.35; p = 0.087). No differences were observed in the proportion of circulating immune T helper cells, cytotoxic T cells, macrophages, neutrophils, natural killer, and dendritic cells in control and MatSep rats. Our findings suggest that MatSep during early life may protect from tubular injury in response to inhibition of nitric oxide synthase. Future studies are warranted to determine whether the observed renal tubular protection is linked to LNAME induced blood pressure elevation, loss of tubular nitric oxide production, and T cell activation.Support or Funding InformationFunded by Cardiovascular Pathophysiology NIH/NHLBI T32HL007918‐19 to IEO, R01DK059600 to JFG, and P01HL69999 to JSP.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carbon monoxide does not contribute to vascular tonus improvement in exercise-trained rats with chronic nitric oxide synthase inhibition

Carbon monoxide (CO), an end product of heme oxygenase (HO) that is involved in the regulation of vascular tonus, may show a compensatory effect in nitric oxide (NO) deficiency. This study aimed to assess the effect of the HO/CO system on the vascular tone in exercise-trained rats with hypertension induced by chronic NO synthase (NOS) inhibition. Hypertension was induced by N-nitro-l-arginine methyl ester (25 mg/kg/day in drinking water), and exercise training comprised swimming 1 h/day, 5 days/week, for 6 weeks. Systolic blood pressure (BP) was measured weekly using a tail-cuff method. The effects of hypertension and/or exercise-training on the constriction and relaxation responses of the thoracic aorta and resistance arteries of the mesenteric and gastrocnemius vascular beds were evaluated. NOS inhibition produced a gradually developed hypertension, and the magnitude of the increase in BP was significantly attenuated by exercise training. Although phenylephrine (Phe)-induced contraction responses of aorta incubated with an HO-1 inhibitor were reduced in hypertensive animals, there was no difference in the hypertensive-exercise group. However, thoracic aortas in the hypertensive-exercise group exhibited significantly more relaxation in response to a CO donor. There was no change in Phe-induced contraction with or without HO inhibition CO donor relaxation responses in both resistance arteries. These results suggest that the HO/CO system does not contribute to diminishing BP by exercise training in a NOS inhibition-induced hypertension model.

Effects of chronic nitric oxide synthase inhibition on V’O2max and exercise capacity in mice

Acute inhibition of NOS by L-NAME (Nω-nitro-L-arginine methyl ester) is known to decrease maximal oxygen consumption (V'O2max) and impair maximal exercise capacity, whereas the effects of chronic L-NAME treatment on V'O2max and exercise performance have not been studied so far. In this study, we analysed the effect of L-NAME treatment, (LN2 and LN12, respectively) on V'O2max and exercise capacity (in maximal incremental running and prolonged sub-maximal incremental running tests), systemic NO bioavailability (plasma nitrite (NO2-) and nitrate (NO3-)) and prostacyclin (PGI2) production in C57BL6/J mice. Mice treated with L-NAME for 2weeks (LN2) displayed higher V'O2max and better running capacity than age-matched control mice. In LN2 mice, NO bioavailability was preserved, as evidenced by maintained NO2- plasma concentration. PGI2 production was activated (increased 6-keto-PGF1α plasma concentration) and the number of circulating erythrocytes (RBC) and haemoglobin concentration were increased. In mice treated with L-NAME for 12weeks (LN12), NO bioavailability was decreased (lower NO2- plasma concentration), and 6-keto-PGF1α plasma concentration and RBC number were not elevated compared to age-matched control mice. However, LN12 mice still performed better during the maximal incremental running test despite having lower V'O2max. Interestingly, the LN12 mice showed poorer running capacity during the prolonged sub-maximal incremental running test. To conclude, short-term (2weeks) but not long-term (12weeks) treatment with L-NAME activated robust compensatory mechanisms involving preservation of NO2- plasma concentration, overproduction of PGI2 and increased number of RBCs, which might explain the fully preserved exercise capacity despite the inhibition of NOS.

Systemic Aldosterone, But Not Angiotensin II, Plays a Pivotal Role in the Pathogenesis of Renal Injury in Chronic Nitric Oxide-Deficient Male Rats

Chronic inhibition of nitric oxide synthase by N(ω)-nitro-L-arginine methyl ester (L-NAME) causes progressive renal injury and systemic hypertension. Angiotensin II (Ang II) has been conventionally regarded as one of the primary causes of renal injury. We reported previously that such renal injury was almost completely suppressed by both an Ang II type I receptor blocker and an aldosterone antagonist. The aldosterone antagonist also inhibited the systemic Ang II elevation. Therefore, it remains to be elucidated whether Ang II or aldosterone directly affects the development of such renal injury. In the present study, we investigated the role of aldosterone in the pathogenesis of renal injury induced by L-NAME-mediated chronic nitric oxide synthase inhibition in male Wistar rats (aged 10 wk). Serial analyses demonstrated that the renal injury and inflammation in L-NAME-treated rats was associated with elevation of both Ang II and aldosterone. To investigate the direct effect of aldosterone on the renal injury, we conducted adrenalectomy (ADX) and aldosterone supplementation in L-NAME-treated rats. In ADX rats, aldosterone was undetectable, and renal injury and inflammation were almost completely prevented by ADX, although systemic and local Ang II and blood pressure were still elevated. Aldosterone supplementation reversed the beneficial effect of ADX. The present study indicates that aldosterone rather than Ang II plays a central and direct role in the pathogenesis of renal injury by L-NAME through inflammation, independent of its systemic hemodynamic effects.

The optimization of a chronic nitric oxide synthase (NOS) inhibition model of pre-eclampsia by evaluating physiological changes

ObjectivesIn order to address the gap in our understanding of the pathogenesis and pathophysiology of PE, we optimized the NOS inhibition animal model by comparing changes in different parameters at various time frames during pregnancy, in both early and late-onset PE. Study Design120 nulliparous Sprague-Dawley rats were divided into 5 groups (n=24). A pregnant control, two groups that represented early and late-onset PE and two groups that were treated with sildenafil citrate (SC) to show reversal of the pre-eclamptic-like symptoms. ResultsOur results showed that treatment with l-NAME caused significant changes in physiological parameters for both early and late-onset PE groups. There was a significant increase in systolic blood pressure (SBP) levels in the early-onset PE group (128.5±5.71mmHg) and late-onset PE group (128.3±6.15mmHg) on day 19 compared to the SBPs on day 0, (p<0.01). Urine excretion volumes in the early-onset PE group (13.62±3.18mL) and in the late-onset PE (13.28±2.60mL), compared to the pregnant control group (11.96±1.9mL) were also increased (p<0.05). There was also an increase in total urinary protein in the early-onset PE group (0.62±0.08g/L and the late-onset PE group (0.45±0.05g/L), when compared to the pregnant control group (0.38±0.07) (p<0.05). We also found a decrease in fetal numbers in the PE group in comparison to the pregnant control and SC treated groups. The remission of these signs was seen after delivery of the fetuses. We also demonstrated that treatment of this syndrome with SC prevented the development of these signs. ConclusionsThe NOS inhibition model can be used for the study of the pathogenesis and pathophysiology of PE, since the pathogenic changes mimic those of early and late-PE.

Chapter 12 - Chronic nitric oxide synthase inhibition does not impair upper airway muscle adaptation to chronic intermittent hypoxia in the rat

Nitric oxide (NO) is an important modulator of striated muscle function. Nitric oxide synthase (NOS) expression and activity is altered by hypoxia and NO is implicated in respiratory muscle remodeling following chronic sustained hypoxia. We sought to determine if NO is implicated in upper airway dilator muscle adaptation to chronic intermittent hypoxia (CIH). Thirty-two adult male Wistar rats (284±13, mean±SD) were exposed to alternating bouts of hypoxia (90 s; 5% O2 at the nadir) and normoxia (210 s; 21% O2) for 12 cycles per hour, 8h/day for 3 weeks. Sham animals were exposed to normoxia in parallel. Half of the animals in both groups received the nNOS inhibitor-L-NNA (2mM) in the drinking water throughout the study (N=8 for all groups). Sternohyoid (pharyngeal dilator) muscle contractile and endurance properties were determined ex vivo. Sternohyoid muscle myosin heavy chain (MHC) isoform composition and cross-sectional area was determined by fluorescence microscopy. Chronic nNOS blockade did not alter sternohyoid muscle peak force or force-frequency relationship in sham or CIH-treated animals. In contrast, chronic nNOS blockade significantly decreased sternohyoid muscle endurance with equivalent effects in sham and CIH-treated rats. Our results suggest that NO is an important modulator of sternohyoid muscle endurance. However, our data provide no evidence to suggest that NO is implicated in upper airway muscle adaptation to CIH.

Reverse myocardial effects of intermedin in pressure-overloaded hearts: role of endothelial nitric oxide synthase activity.

Intermedin (IMD) is a cardiac peptide synthesized in a prepro form, which undergoes a series of proteolytic cleavages and amidations to yield the active forms of 47 (IMD(1-47)) and 40 amino acids (IMD(8-47)). There are several lines of evidence of increased IMD expression in rat models of cardiac pathologies, including congestive heart failure and ischaemia; however, its myocardial effects upon cardiac disease remain unexplored. With this in mind, we investigated the direct effects of increasing concentrations of IMD(1-47) (10(-10) to10(-6) m) on contraction and relaxation of left ventricular (LV) papillary muscles from two rat models of chronic pressure overload, one induced by transverse aortic constriction (TAC), the other by nitric oxide (NO) deficiency due to chronic NO synthase inhibition (NG-nitro-l-arginine, l-NAME), and respective controls (Sham and Ctrl). In TAC and l-NAME rats, exogenous administration of IMD(1-47) elicited concentration-dependent positive inotropic and lusitropic effects. By contrast, in Sham and Ctrl rats, IMD(1-47) induced a negative inotropic response without a significant effect on relaxation. Both TAC and l-NAME rats presented LV hypertrophy, elevated LV systolic pressures, preserved systolic function and elevated peroxynitrite levels. In the normal myocardium (Ctrl and Sham), IMD(1-47) induced a 3-fold increase of endothelial nitric oxide synthase (eNOS) phosphorylation at Ser(1177), indicating enhanced eNOS activity. In TAC and l-NAME rats, eNOS phosphorylation was increased at baseline, and its response to IMD(1-47) was blunted. In addition, the distinct myocardial response to IMD(1-47) was accompanied by distinct subcellular mechanisms. While in Sham rats the addition of IMD(1-47) induced the phosphorylation of cardiac troponin I due to NO/cGMP activation, in TAC rats IMD(1-47) induced phospholamban phosphorylation possibly associated with cAMP/protein kinase A activation. Therefore, we demonstrated for the first time a reversed myocardial response to IMD(1-47) neurohumoral stimulation due to impairment of eNOS activation in TAC and l-NAME rats. These results not only reveal the distinct myocardial effects and subcellular mechanisms for IMD(1-47) in normal and hypertrophic hearts, but also highlight the potential pathophysiological relevance of cardiac endothelial dysfunction in neurohumoral myocardial action.

Protective Effect of Aerobic Exercise against Some of Proinflammatory Cytokines-Induced Chronic Nitric Oxide Synthase Inhibition in Renal Tissue Rats

Protective Effect of Aerobic Exercise against Some of Proinflammatory Cytokines-Induced Chronic Nitric Oxide Synthase Inhibition in Renal Tissue Rats

Abstract 440: Female Spontaneously Hypertensive Rats are More Dependent on Nitric Oxide in Modulating Blood Pressure and Renal Injury Than Males

We recently demonstrated that female spontaneously hypertensive rats (SHR) have higher nitric oxide (NO) bioavailability in the kidney compared to males. Therefore, we hypothesize that female SHR are more dependent on the NO synthase (NOS) system to modulate their blood pressure, and as a result will have a greater rise in blood presure and renal injury in response to chronic NOS inhibition compared to males. Mean arterial pressure in SHR was very sensitive to NOS inhibition, with both sexes exhibiting a significant increase in blood pressure to the non-specific NOS inhibitor N G -nitro-L-arginine methyl ester (L-NAME) at a dose of 2 mg/kg/day. However, there was not a sex difference in the percent increase in mean arterial pressure. Treatment of female and male SHR with 7 mg/kg/day L-NAME for 2 weeks significantly increased mean arterial pressure and indices of renal injury in both females and males; the percent increases in mean arterial pressure and injury were greater in females during the last three days of L-NAME treatment (% increase in mean arterial pressure was 44± 1.3 in females vs. 35± 2.9 in males, P&lt;0.05). L-NAME treatment also increased indices of inflammation and oxidative stress as urinary monocyte chemoattractant protein-1 (MCP-1) and thiobarbituric acid reactive substances (TBARs) excretion levels were increased by L-NAME; however, the increases were greater in females. Renal cortical macrophage and T cell infiltrations and soluble intracellular adhesion molecule-1 (sICAM-1) were also elevated following L-NAME treatment (cortical sICAM-1 levels increased from 4.7± 0.2 to 9± 0.1 ng/mg in males vs. 4.6± 0.1 to 7.9± 0.3 ng/mg in females, P&lt;0.05). Although renal cortical macrophage infiltration was greater in female SHR vs. males following L-NAME treatment, the increase in cortical T cell infiltration and sICAM-1 were greater in males. These data suggest that the NO system plays an important role in modulating blood pressure, renal injury and inflammation in SHR, with females being more dependent on the NOS system than males.

Role of Propolis on Tyrosine Hydroxylase Activity and Blood Pressure in Nitric Oxide Synthase-Inhibited Hypertensive Rats

Reduction in the synthesis or bioavailability of nitric oxide plays a significant role in the development of hypertension. Propolis is a resinous product collected by honeybees from various plant sources. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines. The aim of this study was to examine the effect of propolis on blood pressure (BP), TH, and total RNA levels in the adrenal medulla, heart, and hypothalamus tissues in chronic nitric oxide synthase (NOS)-inhibited rats by Nw-nitro-l-arginine methyl ester (L-NAME). Rats received NOS inhibitor (L-NAME) for 15 days to produce hypertension and propolis for the last 5 days. TH activity and total RNA levels significantly increased in adrenal medulla, heart, and hypothalamus tissues in L-NAME-treated groups (P < .05). TH activity and total RNA levels of L-NAME+propolis-treated rats reduced (P < .05) compared with L-NAME-treated groups. TH activity in propolis-treated rats was reduced to the control values. L-NAME led to a significant increase in BP compared with the control group. Propolis administration to L-NAME-treated rats reduced BP but this was not statistically significant compared to L-NAME-treated groups. These results suggest that propolis decreases TH activity in NOS-inhibited hypertensive rats and thereby may modulate the synthesis of catecholamine and BP.

Protective actions of nebivolol on chronic nitric oxide synthase inhibition-induced hypertension and chronic kidney disease in the rat: a comparison with angiotensin II receptor blockade

Nitric oxide (NO) deficiency contributes to chronic kidney disease (CKD) progression and hypertension. The β-blocker, nebivolol (N), also enhances NO production, and we studied whether N attenuates CKD and hypertension caused by chronic NO synthase inhibition (CNOSI). Male Sprague-Dawley rats on 6 weeks of CNOSI (L-NAME, 150 mg/L drinking water) received placebo (P), N (10 mg/kg/day), olmesartan (O, 2.5 mg/kg/day) or N + O. Blood pressure (BP) and urine protein and NOx (metabolites of NO) were monitored throughout. We measured glomerular sclerosis (GS), creatinine clearance (C(Cr)) and components of the NO and oxidant pathways in the renal cortex. BP increased >50 mmHg in P by weeks 4-6, but no change occurred in N, O or N + O. P rats developed proteinuria and GS and C(Cr) was ∼30% of normal. In N, O and N + O, all values remained normal. In renal cortex of P, p22phox and nitrotyrosine abundance as well as H(2)O(2) levels were higher and extracellular superoxide dismutase (EC SOD) was lower versus normal kidneys. N, O and N + O normalized p22phox, H(2)O(2) and EC SOD and increased Mn SOD above normal. The cortical neuronal NO synthase (nNOS) β abundance increased in P and this was prevented by N, O and N + O. We suggest that the major benefit from both N and O is reduction in oxidative stress in the renal cortex, which may potentiate residual local NO. There was no additive benefit of N + O since each drug effectively prevented injury, but a combination may be beneficial where protection is incomplete with each drug. The increased nNOSβ protein seen early in the course of the CKD may contribute to the evolving GS.