Effects of direct high sodium exposure at endothelial cell migration.

The present study aimed to test the hypothesis that high sodium affects the migratory phenotype of endothelial cells (EC) and investigates mechanisms involved independently of hemodynamic factors. Cell migration was evaluated by Wound-Healing at conditions: High Sodium (HS; 160 mM) and Control (CT; 140 mM). O2- production was evaluated by DHE. NADPH oxidase activity was determined by chemiluminescence assay. Expression of adhesion molecules was analyzed by RT-PCR. Shear Stress was performed using a rhythmic shake. Nitric oxide production was measured by Griess reaction. HS-induced impairment in EC migration while both Candesartan and DPI prevented it. HS increased NADPH oxidase activity, which was blocked by Candesartan. Also, HS increased O2- production that was inhibited by Candesartan. HS decreased adhesion molecules expression via ROS (Integrin Alpha 5, Integrin Beta 1, Integrin Beta 3, VE-Cadherin and PECAM) and via AT1R (PECAM). The nitric oxide production induced by shear stress was decreased after EC exposure to HS while both Candesartan and DPI prevented it. Conclusion: This study demonstrated that HS reduced EC migration by AT1R and ROS derived from NADPH Oxidase and mitochondria. The HS reduction in adhesion molecules expression modulated by ROS and AT1R may help to explain the impairment in migration capacity. Also, HS affected EC functionality by reducing their nitric oxide production in response to shear stress.

Antioxidant effect of sildenafil: Potential hepatoprotection via differential expression of mitochondrial proteins in apolipoprotein E knockout mice.

BACKGROUND: High plasma cholesterol levels are able to trigger several pathophysiological events, including inflammation, cell damage and especially oxidative stress. Previously, studies have shown that sildenafil exhibited antioxidant effects in several experimental models. Here we evaluate the role of sildenafil in liver redox equilibrium of apolipoprotein E knockout (apoE-KO) mice. METHODS: ApoE-KO mice were divided in two groups: one group received the PDE5 inhibitor sildenafil (40 mg/kg/day) for 3 weeks (apoE-KO + Sil) and was compared to a second group of apoE-KO mice, which received only the vehicle (water) for 3 weeks (apoE-KO). Control group (C57 mice) received only a standard chow diet. At the age of 18 weeks, mice livers were collected for the measurement of intracellular ROS levels and apoptotic cells by flow cytometry analysis, and mitochondria isolation for proteomic analysis. RESULTS: Compared to the control group, liver cells from apoE-KO presented some typical redox imbalance features: higher levels of intracellular ROS (global oxidative stress ˜60%, superoxide anion ˜82%, and peroxynitrite/hydroxyl radical ˜53%), higher amounts of apoptotic cells (up to ˜19%) and higher mitochondrial intensity of catalase (+339%) and transferrin spots (+914%). After treatment with sildenafil, apoE-KO presented ROS levels and the number of apoptotic cells similar to those observed in C57. In addition, when compared to apoE-KO, apoE-KO + Sil showed lower spots volumes of catalase (-23%) and transferrin (-71%) and up-regulation of urate oxidase (+94%). CONCLUSION: The treatment with sildenafil is able to induce beneficial changes in liver mitochondrial protein dynamics, which restores the redox homeostasis contributing to a potential hepatoprotection.

Increased ROS production and DNA damage in monocytes are biomarkers of aging and atherosclerosis.

BACKGROUND: New evidence demonstrates that aging and dyslipidemia are closely associated with oxidative stress, DNA damage and apoptosis in some cells and extravascular tissues. However, in monocytes, which are naturally involved in progression and/or resolution of plaque in atherosclerosis, this concurrence has not yet been fully investigated. In this study, we evaluated the influence of aging and hypercholesterolemia on serum pro-inflammatory cytokines, oxidative stress, DNA damage and apoptosis in monocytes from apolipoprotein E-deficient (apoE-/-) mice compared with age-matched wild-type C57BL/6 (WT) mice. Experiments were performed in young (2-months) and in old (18-months) male wild-type (WT) and apoE-/- mice. RESULTS: Besides the expected differences in serum lipid profile and plaque formation, we observed that atherosclerotic mice exhibited a significant increase in monocytosis and in serum levels of pro-inflammatory cytokines compared to WT mice. Moreover, it was observed that the overproduction of ROS, led to an increased DNA fragmentation and, consequently, apoptosis in monocytes from normocholesterolemic old mice, which was aggravated in age-matched atherosclerotic mice. CONCLUSIONS: In this study, we demonstrate that a pro-inflammatory systemic status is associated with an impairment of functionality of monocytes during aging and that these parameters are fundamental extra-arterial contributors to the aggravation of atherosclerosis. The present data open new avenues for the development of future strategies with the purpose of treating atherosclerosis.

Beneficial Morphofunctional Changes Promoted by Sildenafil in Resistance Vessels in the Angiotensin II-Induced Hypertension Model.

BACKGROUND: By acting on multiple targets and promoting diverse actions, angiotensin II (Ang II) plays a pivotal role in vascular function. Recent studies suggested that phosphodiesterase-5 (PDE-5) inhibitors exhibit therapeutic effects in cardiovascular diseases. Here, the effects of sildenafil on vascular disturbances were analyzed in a mouse model of Ang II-induced hypertension. METHODS AND RESULTS: Male C57BL/6 mice were used as untreated animals (control) or infused with Ang II (1000 ηg/kg/min) for 28 days and treated with sildenafil (40 mg/kg/min) or vehicle (Ang II) during the last two weeks. After 4 weeks, the Ang II animals exhibited a high systolic blood pressure (186±3 mmHg vs. 127±3 mmHg for control mice), which was attenuated by sildenafil (163±7 mmHg). The mesenteric vessels from the Ang II animals revealed damage to the endothelial layer, an increase in the cross-section area (1.9-fold) and vascular cell production of peroxynitrite (512±13 a.u.), which was ameliorated in the Ang II-Sil group (1.2-fold and 400±17 a.u.). Analysis of the vascular responsiveness showed an increased contractility response to norepinephrine in Ang II animals (Rmax: 70%), which was abolished by sildenafil through increased nitric oxide (NO) bioavailability and decreased reactive oxygen species (ROS) and vasoconstrictor prostanoids. CONCLUSION: Sildenafil attenuates the morphofunctional deleterious effects of Ang II on resistance vessels. The benefits of sildenafil seem to occur through restoring the balance of ROS/NO/eicosanoids. Therefore, this study opened new avenues for further clinical targeting of the treatment of cardiovascular diseases related to activation of the renin-angiotensin system.

Increased ROS production and DNA damage in monocytes are biomarkers of aging and atherosclerosis

BACKGROUND: New evidence demonstrates that aging and dyslipidemia are closely associated with oxidative stress, DNA damage and apoptosis in some cells and extravascular tissues. However, in monocytes, which are naturally involved in progression and/or resolution of plaque in atherosclerosis, this concurrence has not yet been fully investigated. In this study, we evaluated the influence of aging and hypercholesterolemia on serum pro-inflammatory cytokines, oxidative stress, DNA damage and apoptosis in monocytes from apolipoprotein E-deficient (apoE-/-) mice compared with age-matched wild-type C57BL/6 (WT) mice. Experiments were performed in young (2-months) and in old (18-months) male wild-type (WT) and apoE-/- mice. RESULTS: Besides the expected differences in serum lipid profile and plaque formation, we observed that atherosclerotic mice exhibited a significant increase in monocytosis and in serum levels of pro-inflammatory cytokines compared to WT mice. Moreover, it was observed that the overproduction of ROS, led to an increased DNA fragmentation and, consequently, apoptosis in monocytes from normocholesterolemic old mice, which was aggravated in age-matched atherosclerotic mice. CONCLUSIONS: In this study, we demonstrate that a pro-inflammatory systemic status is associated with an impairment of functionality of monocytes during aging and that these parameters are fundamental extra-arterial contributors to the aggravation of atherosclerosis. The present data open new avenues for the development of future strategies with the purpose of treating atherosclerosis.

Sildenafil (Viagra®) Prevents Cox-1/ TXA2 Pathway-Mediated Vascular Hypercontractility in ApoE-/- Mice.

BACKGROUND/AIMS: The atherosclerotic apolipoprotein E-deficient (apoE-/-) mouse exhibits impaired vasodilation and enhanced vasoconstriction responsiveness. The objectives of this study were: a) to determine the relative contribution of cyclooxygenases (Cox-1 and Cox-2), thromboxane A2 (TXA2) and endothelin-1 (ET-1) to enhancing vascular hyperresponsiveness in this model of atherosclerosis and b) to investigate the beneficial effects of the phosphodiesterase 5 inhibitor sildenafil on this endothelial dysfunction. METHODS: Adult male apoE-/- mice were treated with sildenafil (40 mg/kg/day, for 3 weeks) and compared with non-treated ApoE-/- and wild-type mice. The beneficial effects of sildenafil on vascular contractile response to phenylephrine (PE) in aortic rings were evaluated before and after incubation with Cox-1 (SC-560) or Cox-2 (NS-398) inhibitors or the TP antagonist SQ-29548, and on contractile responsiveness to ET-1. RESULTS: ApoE-/- mice exhibited enhanced vasoconstriction to PE (Rmax ∼35%, p<0.01), which was prevented by treatment with sildenafil. The enhanced PE-induced contractions were abolished by both Cox-1 inhibition and TP antagonist, but were not modified by Cox-2 inhibition. Aortic rings from ApoE-/- mice also exhibited enhanced contractions to ET-1 (Rmax ∼30%, p<0.01), which were attenuated in sildenafil-treated ApoE-/- mice. In addition, we observed augmented levels of vascular proinflammatory cytokines in ApoE-/- mice, which were partially corrected by treatment with sildenafil (IL-6, IL-10/IL-6 ratio and MCP-1). CONCLUSION: The present data show that the Cox-1/TXA2 pathway prevails over the Cox-2 isoform in the mediation of vascular hypercontractility observed in apoE-/-mice. The results also show a beneficial effect of sildenafil on this endothelial dysfunction and on the proinflammatory cytokines in atherosclerotic animals, opening new perspectives for the treatment of other endothelium-related cardiovascular abnormalities.

Chronic administration of the soluble, nonbacterial fraction of kefir attenuates lipid deposition in LDLr-/- mice.

OBJECTIVES: Kefir is obtained by the action of acidic bacteria and yeasts that exist in symbiotic association in kefir grains. Recently, this fermented milk drink has been recommended for the treatment of several clinical conditions, such as inflammatory, gastrointestinal, or cardiovascular-related diseases, or a combination of these diseases. However, its effects on atherosclerosis are not yet clear. The aim of this study was to prove that chronic treatment with a soluble, nonbacterial fraction of kefir could reduce the progression of atherosclerosis in low-density lipoprotein receptor-deficient (LDLr-/-) mice. METHODS: LDLr-/- mice were divided into four groups as follows: RESULTS: The soluble, nonbacterial fraction of kefir reduced lipid deposition (P < 0.05) independent of hypercholesterolemia. Moreover, kefir was capable of diminishing the circulating proinflammatory intereukin (IL)-6 level and the ratio of tumor necrosis factor-α to IL-10 (50% and 42%, P < 0.05, respectively) and augmenting the antiinflammatory IL-10 level by approximately 74% (P < 0.05). CONCLUSIONS: Chronic treatment with a soluble nonbacterial fraction of kefir was able to decrease the lipid deposition in LDLr-/- hypercholesteremic mice, at least in part through modifying the circulating cytokine profile. The beneficial effects of kefir provide new perspectives for its use as an adjuvant in the prevention of atherosclerosis.

Increased Blood Pressure Variability Prior to Chronic Kidney Disease Exacerbates Renal Dysfunction in Rats.

Increased blood pressure variability (BPV), which can be experimentally induced by sinoaortic denervation (SAD), has emerged as a new marker of the prognosis of cardiovascular and renal outcomes. Considering that increased BPV can lead to organ-damage, the goal of the present study was to evaluate the effects of SAD on renal function in an experimental model of chronic kidney disease (CKD). SAD was performed in male Wistar rats 2 weeks before 5/6 nephrectomy and the animals were evaluated 4 weeks after the induction of CKD. Our data demonstrated that BPV was increased in SAD and CKD animals and that the combination of both conditions (SAD+CKD) exacerbated BPV. The baroreflex sensitivity index was diminished in the SAD and CKD groups; this reduction was more pronounced when SAD and CKD were performed together. 5/6 nephrectomy led to hypertension, which was higher in SAD+CKD animals. Regarding renal function, the combination of SAD and CKD resulted in reduced renal plasma and blood flow, increased renal vascular resistance and augmented uraemia when compared to CKD animals. Glomerular filtration rate and BPV were negatively correlated in SAD, CKD, and SAD+CKD animals. Moreover, SAD+CKD animals presented a higher level of glomerulosclerosis when compared to all other groups. Cardiac and renal hypertrophy, as well as oxidative stress, was also further increased when SAD and CKD were combined. These results show that SAD prior to 5/6 nephrectomy exacerbates renal dysfunction, suggesting that previous augmented BPV should be considered as an important factor to the progression of renal diseases.

Tributyltin chloride induces renal dysfunction by inflammation and oxidative stress in female rats.

Tributyltin chloride (TBT) is an organometallic pollutant that is used as a biocide in antifouling paints. TBT induces several toxic and endocrine-disrupting effects. However, studies evaluating the effects of TBT on renal function are rare. This study demonstrates that TBT exposure is responsible for improper renal function as well as the development of abnormal morphophysiology in mammalian kidneys. Female rats were treated with TBT, and their renal morphophysiology was assessed. Morphophysiological abnormalities such as decreased glomerular filtration rate and increased proteinuria levels were observed in TBT rats. In addition, increases in inflammation, collagen deposition and α-smooth muscle actin (α-SMA) protein expression were observed in TBT kidneys. A disrupted cellular redox balance and apoptosis in kidney tissue were also observed in TBT rats. TBT rats demonstrated reduced serum estrogen levels and estrogen receptor-α (ERα) protein expression in renal cortex. Together, these data provide in vivo evidence that TBT is toxic to normal renal function and that these effects may be associated with renal histopathology complications, such as inflammation and fibrosis.

Protective effect of sildenafil on the genotoxicity and cytotoxicity in apolipoprotein E-deficient mice bone marrow cells.

BACKGROUND: The pharmacological inhibitor of phosphodiesterase 5 (PDE5), sildenafil, is a promising candidate for antioxidant therapy that can result in cardiovascular protection. In addition to its known effects on the cardiovascular system, hypercholesterolemia leads to increased oxidative stress and DNA damage in the bone marrow, which is a non-classical target organ of atherosclerosis. In the present study, we evaluate oxidative stress and assess the effect of genomic instability on cell cycle kinetics in atherosclerotic animals and determine if sildenafil reverses these detrimental effects in bone marrow cells. METHODS: Experiments were performed in male wild-type (WT) and apolipoprotein E knockout mice (apoE(-/-)) (9 weeks of age). apoE(-/-) mice were randomly distributed into the following 2 groups: sildenafil-treated (40 mg/kg/day for 3 weeks, n = 8) and vehicle-treated (n = 8), by oral gavage. After treatment, bone marrow cells were isolated to assess the production of superoxide anions and hydrogen peroxide, determine cell cycle kinetics and evaluate the presence of micronucleated cells. RESULTS: Sildenafil treatment reduced the cytoplasmic levels of superoxide anion (~95% decrease, p < 0.05) and decreased hydrogen peroxide (~30% decrease, p < 0.05). Moreover, we observed protective effects on the DNA of bone marrow cells, including normal cell cycling, decreased DNA fragmentation and a diminished frequency of micronucleated cells. CONCLUSION: Our data reveal that the excessive production of ROS in atherosclerotic mice overcome the DNA repair pathways in bone marrow cells. The novelty of the present study is that the administration of sildenafil reduced ROS to baseline levels and, consequently, reverted the DNA damage and its outcomes in bone marrow cells.

Predictive value of cystatin C for the identification of illness severity in adult patients in a mixed intensive care unit.

OBJECTIVES: This study compared serum cystatin C (CysC) with conventional biomarkers of renal function in terms of their ability to predict illness severity in patients in a mixed intensive care unit (ICU). The present study also tested the hypothesis that increased CysC could predict illness severity in different clinical conditions in adult patients admitted to the ICU. DESIGN AND METHODS: The performance of serum creatinine, urea and CysC, as well as the Glomerular Filtration Rate (GFR) estimates (Cockcroft-Gault/MDRD/Larsson and CKD-EPI Equations) in predicting illness severity was compared in 60 critically ill patients. Adult patients admitted to the hospital were screened for eligibility in this prospective and observational study. The mean patient age was 52±19years. The average APACHE II score was 9.5±6 for the entire sample. The patients were assigned to two different degrees of severity, and the internally derived cut off value was an APACHE II score<10 or ≥10. RESULTS: Both serum CysC and urea showed significant correlations with APACHE II, even after controlling for age. Urea and CysC levels, as well as the GFR estimated by the method of Larsson and Cockcroft-Gault, remained significantly increased in patients in the APACHEII ≥10 group. The ROC curve analyses indicated that both urea and CysC levels have high sensitivity and specificity in the prediction of illness severity using the APACHE II as a gold standard prognostic stratification system. Furthermore, CysC was more accurate than the Larsson, CKD-EPI CysC, CKD-EPI Cr-CysC, Cockcroft-Gault and CKD-EPI Cr CFR estimation methods compared with the MDRD method. Additionally, CysC was a good predictor in both young and old patients, whereas urea was not predictive of illness severity. CONCLUSIONS: Our findings suggest that CysC and GFR estimates (Larsson or CKD-EPI CysC methods) are good predictors of illness severity in adult patients hospitalized in a mixed ICU.

Chronic administration of the probiotic kefir improves the endothelial function in spontaneously hypertensive rats.

BACKGROUND: The beverage obtained by fermentation of milk with kefir grains, a complex matrix containing acid bacteria and yeasts, has been shown to have beneficial effects in various diseases. However, its effects on hypertension and endothelial dysfunction are not yet clear. In this study, we evaluated the effects of kefir on endothelial cells and vascular responsiveness in spontaneously hypertensive rats (SHR). METHODS: SHR were treated with kefir (0.3 mL/100 g body weight) for 7, 15, 30 and 60 days and compared with non-treated SHR and with normotensive Wistar-Kyoto rats. Vascular endothelial function was evaluated in aortic rings through the relaxation response to acetylcholine (ACh). The balance between reactive oxygen species (ROS) and nitric oxide (NO) synthase was evaluated through specific blockers in the ACh-induced responses and through flow cytometry in vascular tissue. RESULTS: Significant effects of kefir were observed only after treatment for 60 days. The high blood pressure and tachycardia exhibited by the SHR were attenuated by approximately 15 % in the SHR-kefir group. The impaired ACh-induced relaxation of the aortic rings observed in the SHR (37 ± 4 %, compared to the Wistar rats: 74 ± 5 %), was significantly attenuated in the SHR group chronically treated with kefir (52 ± 4 %). The difference in the area under the curve between before and after the NADPH oxidase blockade or NO synthase blockade of aortic rings from SHR were of approximately +90 and -60 %, respectively, when compared with Wistar rats. In the aortic rings from the SHR-kefir group, these values were reduced to +50 and -40 %, respectively. Flow cytometric analysis of aortic endothelial cells revealed increased ROS production and decreased NO bioavailability in the SHR, which were significantly attenuated by the treatment with kefir. Scanning electronic microscopy showed vascular endothelial surface injury in SHR, which was partially protected following administration of kefir for 60 days. In addition, the recruitment of endothelial progenitor cells was decreased in the non-treated SHR and partially restored by kefir treatment. CONCLUSIONS: Kefir treatment for 60 days was able to improve the endothelial function in SHR by partially restoring the ROS/NO imbalance and the endothelial architecture due to endothelial progenitor cells recruitment.

Reactive oxygen species contribute to dysfunction of bone marrow hematopoietic stem cells in aged C57BL/6 J mice.

BACKGROUND: Stem cells of intensely regenerative tissues are susceptible to cellular damage. Although the response to this process in hematopoietic stem cells (HSCs) is crucial, the mechanisms by which hematopoietic homeostasis is sustained are not completely understood. Aging increases reactive oxygen species (ROS) levels and inflammation, which contribute to increased proliferation, senescence and/or apoptosis, leading to self-renewal premature exhaustion. In this study, we assessed ROS production, DNA damage, apoptosis, senescence and plasticity in young, middle and aged (2-, 12- and 24-month-old, respectively) C57BL/6 J mice. RESULTS: Aged HSCs showed an increase in intracellular superoxide anion (1.4-fold), hydrogen peroxide (2-fold), nitric oxide (1.6-fold), peroxynitrite/hidroxil (2.6-fold) compared with young cells. We found that mitochondria and NADPHox were the major sources of ROS production in the three groups studied, whereas CYP450 contributed in middle and aged, and xanthine oxidase only in aged HSCs. In addition, we observed DNA damage and apoptosis in the middle (4.2- and 2-fold, respectively) and aged (6- and 4-fold, respectively) mice; aged mice also exhibited a significantly shorter telomere length (-1.8-fold) and a lower expression of plasticity markers. CONCLUSION: These data suggest that aging impairs the functionality of HSCs and that these age-associated alterations may affect the efficacy of aged HSC recovery and transplantation.

The protective effects of oral low-dose quercetin on diabetic nephropathy in hypercholesterolemic mice.

AIMS: Diabetic nephropathy (DN) is one of the most important causes of chronic renal disease, and the incidence of DN is increasing worldwide. Considering our previous report (Gomes et al., 2014) indicating that chronic treatment with oral low-dose quercetin (10 mg/Kg) demonstrated anti-oxidative, anti-apoptotic and renoprotective effects in the C57BL/6J model of DN, we investigated whether this flavonoid could also have beneficial effects in concurrent DN and spontaneous atherosclerosis using the apolipoprotein E-deficient mouse (apoE(-/-)). METHODS: Streptozotocin was used to induce diabetes (100 mg/kg/day, 3 days) in male apoE(-/-) mice (8 week-old). After 6 weeks, the mice were randomly separated into DQ: diabetic apoE(-/-) mice treated with quercetin (10 mg/kg/day, 4 weeks, n = 8), DV: diabetic ApoE(-/-) mice treated with vehicle (n = 8) and ND: non-treated non-diabetic mice (n = 8). RESULTS: Quercetin treatment diminished polyuria (~30%; p < 0.05), glycemia (~25%, p < 0.05), normalized the hypertriglyceridemia. Moreover, this bioflavonoid diminished creatininemia (~30%, p < 0.01) and reduced proteinuria but not to normal levels. We also observed protective effects on the renal structural changes, including normalization of the index of glomerulosclerosis and kidney weight/body weight. CONCLUSIONS: Our data revealed that quercetin treatment significantly reduced DN in hypercholesterolemic mice by inducing biochemical changes (decrease in glucose and triglycerides serum levels) and reduction of glomerulosclerosis. Thus, this study highlights the relevance of quercetin as an alternative therapeutic option for DN, including in diabetes associated with dyslipidemia.

Increased oxidative stress and apoptosis in peripheral blood mononuclear cells of fructose-fed rats.

BACKGROUND: Measuring of oxidative stress in peripheral blood mononuclear cells is a suitable model of dietary induced systemic oxidative stress. Thus, we aimed to evaluate whether a chronic high fructose intake could induce oxidative damage in peripheral blood and bone marrow mononuclear cells of rats. METHODS: Animals were randomly assigned to the following groups: Control group (standard rat chow and tap water n=8), and Fructose group (standard rat chow and a 10% fructose solution in the drinking water n=8). Reactive oxygen species and cytokines were measure using flow cytometry in peripheral blood and bone-marrow mononuclear cells. Apoptotic cell death and the advanced oxidation protein products (AOPP) were also determined. RESULTS: We observed a significant increase in ROS production in peripheral blood mononuclear cells of fructose group as compared to control rats. Apoptosis and the AOPP were higher in those animals underwent high fructose intake. Serum levels of IL-6 and IL-12 were also increased after 12 weeks of high fructose intake. CONCLUSION: We concluded that fructose intake leads to systemic oxidative stress and pro-inflammatory condition which affect peripheral blood mononuclear cells and bone-marrow mononuclear cells viability.

Sildenafil Improves Vascular Endothelial Structure and Function in Renovascular Hypertension.

In translational medicine, the discovery of new drugs or new potential uses for currently available drugs is crucial for treating the resistant hypertension associated with renal artery stenosis. The phosphodiesterase 5 inhibitor sildenafil has been shown to reduce blood pressure and to improve the endothelium-dependent relaxation in the two kidney, one clip (2K1C) mouse model of renovascular hypertension. In the present study, we evaluated the effects of sildenafil (40 mg/kg/day for two weeks) on the endothelial structure and contractile function in mesenteric resistance arteries 28 days after clipping the renal artery. The data showed an enhanced vascular contractile response to norepinephrine in 2K1C hypertensive mice (56%) when compared with Sham mice, which was associated with increased oxidative stress and with a thinning of endothelial cells. Sildenafil treatment caused a significant amelioration in the enhanced contractile responsiveness (18%), which was associated to the recovery of the endothelial surface and abolishment of the oxidative stress. These data suggest that sildenafil could be considered a promising therapeutic option to manage endothelial dysfunction and hypertension in resistant patients.

Mechanisms of enhanced vasoconstriction in the mouse model of atherosclerosis: the beneficial effects of sildenafil.

Sildenafil ameliorates aortic relaxations in apolipoprotein E knockout (apoE) mice. Now, we tested the hypothesis that endothelial dysfunction (ED) in this model is characterized by contractile hyperresponsiveness to phenylephrine (PE) and that this abnormality may be repaired using sildenafil. The aortic rings were evaluated in apoE mice treated with sildenafil (apoE-sil, 40 mg/kg/day) and compared with apoE and wild-type (WT) mice administered with vehicle (veh). The apoE-veh mice exhibited an imbalance of nitric oxide and reactive oxygen species (NO/ROS) levels and an increased maximum response (Rmax, 20%) and sensitivity (7%) to PE, which were not modified by endothelial removal. Under the prostanoids blockade, vasocontraction was decreased more in apoE-veh (-37%) than in WT (-27%) and apoE-sil (-30%) mice. NADPH-oxidase blockade abolished the enhanced contractile responsiveness in apoE-veh (-33%), without effects in WT and apoE-sil groups. The atherosclerotic lesions and the imbalance of NO/ROS were reduced (40%) in apoE-sil mice. In conclusion, ED in apoE mice was characterized by decreased NO-bioavailability and contractile hyperresponsiveness, due to thromboxane and oxidative stress, and was normalized by sildenafil. The beneficial effects of this phosphodiesterase-5 inhibitor on ED and lipid deposition provide new insights for its use as adjuvant in the treatment of atherosclerosis.

Dioclea violacea lectin ameliorates oxidative stress and renal dysfunction in an experimental model of acute kidney injury.

Acute kidney injury (AKI) is characterized by rapid and potentially reversible decline in renal function; however, the current management for AKI is nonspecific and associated with limited supportive care. Considering the need for more novel therapeutic approaches, we believe that lectins from Dioclea violacea (Dvl), based on their anti-inflammatory properties, could be beneficial for the treatment of AKI induced by renal ischemia/reperfusion (IR). Dvl (1 mg/kg, i.v.) or vehicle (100 µL) was administered to Wistar rats prior to the induction of bilateral renal ischemia (45 min). Following 24 hours of reperfusion, inulin and para-aminohippurate (PAH) clearances were performed to determine glomerular filtration rate (GFR), renal plasma flow (RPF), renal blood flow (RBF) and renal vascular resistance (RVR). Renal inflammation was assessed using myeloperoxidase (MPO) activity. Kidney sections were stained with hematoxylin-eosin to evaluate morphological changes. Intracellular superoxide anions, hydrogen peroxide, peroxynitrite, nitric oxide and apoptosis were analyzed using flow cytometry. IR resulted in diminished GFR, RPF, RBF, and increased RVR; however, these changes were ameliorated in rats receiving Dvl. AKI-induced histomorphological changes, such as tubular dilation, tubular necrosis and proteinaceous casts, were attenuated by Dvl administration. Treatment with Dvl resulted in diminished renal MPO activity, oxidative stress and apoptosis in rats submitted to IR. Our data reveal that Dvl has a protective effect in the kidney, improving renal function after IR injury, probably by reducing neutrophil recruitment and oxidative stress. These results indicate that Dvl can be considered a new therapeutic approach for AKI-induced kidney injury.

Renoprotective, anti-oxidative and anti-apoptotic effects of oral low-dose quercetin in the C57BL/6J model of diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is one of the major causes of end-stage renal disease in diabetic patients. Increasing evidence from studies in the rodents has suggested that this disease is associated with increased oxidative stress due to hyperglycemia. In the present study, we evaluated the renoprotective, anti-oxidative and anti-apoptotic effects of the flavonoid quercetin in C57BL/6J model of DN. METHODS: DN was induced by streptozotocin (STZ, 100 mg/kg/day, for 3 days) in adult C57BL/6J mice. Six weeks later, mice were divided into the following groups: diabetic mice treated with quercetin (DQ, 10 mg/kg/day, 4 weeks), diabetic mice treated with vehicle (DV) or non-treated non-diabetic (ND) mice. RESULTS: Quercetin treatment caused a reduction in polyuria (~45%) and glycemia (~35%), abolished the hypertriglyceridemia and had significant effects on renal function including, decreased proteinuria and high plasma levels of uric acid, urea and creatinine, which were accompanied by beneficial effects on the structural changes of the kidney including glomerulosclerosis. Flow cytometry showed a decrease in oxidative stress and apoptosis in DN mice. CONCLUSION: Taken together, these data show that quercetin effectively attenuated STZ-induced cytotoxicity in renal tissue. This study provides convincing experimental evidence and perspectives on the renoprotective effects of quercetin in diabetic mice and outlines a novel therapeutic strategy for this flavonoid in the treatment of DN.

Sildenafil ameliorates oxidative stress and DNA damage in the stenotic kidneys in mice with renovascular hypertension.

BACKGROUND: Oxidative stress and DNA damage have been implicated in the pathogenesis of renovascular hypertension induced by renal artery stenosis in the two-kidney, one-clip (2K1C) Goldblatt model. Considering our previous report indicating that the chronic blockade of phosphodiesterase 5 with sildenafil (Viagra) has marked beneficial effects on oxidative stress and DNA damage, we tested the hypothesis that sildenafil could also protect the stenotic kidneys of 2K1C hypertensive mice against oxidative stress and genotoxicity. METHODS: The experiments were performed with C57BL6 mice subjected to renovascular hypertension by left renal artery clipping. Two weeks after clipping, the mice were treated with sildenafil (40 mg/kg/day for 2 weeks, 2K1C-sildenafil group) or the vehicle (2K1C). These mice were compared with control mice not subjected to renal artery clipping (Sham). After hemodynamic measurements, the stenotic kidneys were assessed using flow cytometry to evaluate cell viability and the comet assay to evaluate DNA damage. Measurements of intracellular superoxide anions and hydrogen peroxide levels as well as nitric oxide bioavailability were also obtained. RESULTS: Sildenafil treatment significantly reduced mean arterial pressure (15%), heart rate (8%), intrarenal angiotensin II (50%) and renal atrophy (36%). In addition, it caused a remarkable decrease of reactive oxygen species production. On the other hand, sildenafil increased nitric oxide levels relative to those in the nontreated 2K1C mice. Sildenafil treatment also significantly reduced the high level of kidney DNA damage that is a characteristic of renovascular hypertensive mice. CONCLUSIONS: Our data reveal that sildenafil has a protective effect on the stenotic kidneys of 2K1C mice, suggesting a new use of phosphodiesterase 5 inhibitors for protection against the DNA damage observed in the hypoperfused kidneys of individuals with renovascular hypertension. Further translational research is necessary to delineate the mechanisms involved in the prevention of renal stenosis in the clinical setting.