The caveolin-1 regulated protein follistatin protects against diabetic kidney disease.

Glomerular matrix protein accumulation, mediated largely by mesangial cells, is central to the pathogenesis of diabetic kidney disease. Our previous studies showed that the membrane microdomains caveolae and their marker protein caveolin-1 regulate matrix protein synthesis in mesangial cells in response to diabetogenic stimuli, and that caveolin-1 knockout mice are protected against diabetic kidney disease. In a screen to identify the molecular mechanism underlying this protection, we also established that secreted antifibrotic glycoprotein follistatin is significantly upregulated by caveolin-1 deletion. Follistatin potently neutralizes activins, members of the transforming growth factor-ß superfamily. A role for activins in diabetic kidney disease has not yet been established. Therefore, in vitro, we confirmed the regulation of follistatin by caveolin-1 in primary mesangial cells and showed that follistatin controls both basal and glucose-induced matrix production through activin inhibition. In vivo, we found activin A upregulation by immunohistochemistry in both mouse and human diabetic kidney disease. Importantly, administration of follistatin to type 1 diabetic Akita mice attenuated early diabetic kidney disease, characterized by albuminuria, hyperfiltration, basement membrane thickening, loss of endothelial glycocalyx and podocyte nephrin, and glomerular matrix accumulation. Thus, activin A is an important mediator of high glucose-induced profibrotic responses in mesangial cells, and follistatin may be a potential novel therapy for the prevention of diabetic kidney disease.

Follistatin Protects Against Glomerular Mesangial Cell Apoptosis and Oxidative Stress to Ameliorate Chronic Kidney Disease.

Aims: Interventions to inhibit oxidative stress and apoptosis, important pathogenic contributors toward the progression of chronic kidney disease (CKD), are not well established. Here, we investigated the role of a transforming growth factor beta (TGFß) superfamily neutralizing protein, follistatin (FST), in the regulation of apoptosis and oxidative stress in glomerular mesangial cells (MCs) and in the progression of CKD. Results: The endoplasmic reticulum (ER) stress inducer thapsigargin (Tg), known to cause MC apoptosis, led to a post-translational increase in the expression of FST. Recombinant FST protected, whereas FST downregulation augmented, Tg-induced apoptosis without affecting Ca2+ release or ER stress induction. Although activins are the primary ligands neutralized by FST, their inhibition with neutralizing antibodies did not affect Tg-induced apoptosis. Instead, FST protected against Tg-induced apoptosis through neutralization of reactive oxygen species (ROS) independently of its ability to neutralize activins. Importantly, administration of FST to mice with CKD protected against renal cell apoptosis and oxidative stress. This was associated with improved kidney function, reduced albuminuria, and attenuation of fibrosis. Innovation and Conclusion: Independent of its activin neutralizing ability, FST protected against Tg-induced apoptosis through neutralization of ROS and consequent suppression of oxidative stress, seen both in vitro and in vivo. Importantly, FST also ameliorated fibrosis and improved kidney function in CKD. FST is, thus, a novel potential therapeutic agent for delaying the progression of CKD. Antioxid. Redox Signal. 31, 551-571.

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.

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.

Effects of Kefir on the Cardiac Autonomic Tones and Baroreflex Sensitivity in Spontaneously Hypertensive Rats.

AIMS: It has been previously shown that the probiotic kefir (a symbiotic matrix containing acid bacteria and yeasts) attenuated the hypertension and the endothelial dysfunction in spontaneously hypertensive rats (SHR). In the present study, the effect of chronic administration of kefir on the cardiac autonomic control of heart rate (HR) and baroreflex sensitivity (BRS) in SHR was evaluated. METHODS: SHR were treated with kefir (0.3 mL/100 g body weight) for 60 days and compared with non-treated SHR and with normotensive Wistar-Kyoto rats. Cardiac autonomic vagal (VT) and sympathetic (ST) tones were estimated through the blockade of the cardiac muscarinic receptors (methylatropine) and the blockade of ß1-adrenoceptor (atenolol). The BRS was evaluated by the tachycardia and bradycardia responses to vasoactive drug-induced decreases and increases in arterial blood pressure (BP), respectively. Additionally, spontaneous BRS was estimated by autoregressive spectral analysis. RESULTS: Kefir-treated SHR exhibited significant attenuation of basal BP, HR, and cardiac hypertrophy compared to non-treated SHR (12, 13, and 21%, respectively). Cardiac VT and ST were significantly altered in the SHR (~40 and ~90 bpm) compared with Wistar rats (~120 and ~30 bpm) and were partially recovered in SHR-kefir (~90 and ~25 bpm). SHR exhibited an impaired bradycardic BRS (~50%) compared with Wistar rats, which was reduced to ~40% in the kefir-treated SHR and abolished by methylatropine in all groups. SHR also exhibited a significant impairment of the tachycardic BRS (~23%) compared with Wistar rats and this difference was reduced to 8% in the SHR-kefir. Under the action of atenolol the residual reflex tachycardia was smaller in SHR than in Wistar rats and kefir attenuated this abnormality. Spectral analysis revealed increased low frequency components of BP (~3.5-fold) and pulse interval (~2-fold) compared with Wistar rats and these differences were reduced by kefir-treatment to ~1.6- and ~1.5-fold, respectively. Spectral analysis also showed an impairment of spontaneous BRS in SHR, but kefir-treatment caused only a tendency to reverse this result. CONCLUSIONS: The novelty of this study is that daily chronic consumption of a low dose of kefir reduced the impairment of the cardiac autonomic control of HR and of the impaired BRS in SHR.

Novel Therapeutic Targets for Phosphodiesterase 5 Inhibitors: current state-of-the-art on systemic arterial hypertension and atherosclerosis.

The usefulness of selective inhibitors of phosphodiesterase 5 (PDE5) is well known, first for the treatment of male erectile dysfunction and more recently for pulmonary hypertension. The discovery that PDE5 is present in the systemic artery endothelium and smooth muscle cells led investigators to test the extra sexual effects of sildenafil, the first and most investigated PDE5 inhibitor, in diseases affecting the systemic arteries. Cumulative data from experimental and clinical studies have revealed beneficial effects of sildenafil on systemic arterial hypertension and its target organs, such as the heart, kidneys and vasculature. An important effect of sildenafil is reduction of hypertension and improvement of endothelial function in experimental models of hypertension and hypertensive subjects. Interestingly, in angiotensin-dependent hypertension, its beneficial effects on endothelial and kidney dysfunctions seem to at least in part be caused by its ability to decrease the levels of angiotensin II and increase angiotensin 1-7, in addition to improving nitric oxide bioavailability and diminishing reactive oxygen species. Another remarkable finding on the effects of sildenafil comes from studies in apolipoprotein E knockout mice, a model of atherosclerosis that closely resembles human atherosclerotic disease. In this review, we focus on the promising beneficial effects of sildenafil for treating systemic high blood pressure, especially resistant hypertension, and the endothelial dysfunction that is present in hypertension and atherosclerosis.

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.

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.

Tributyltin chloride leads to adiposity and impairs metabolic functions in the rat liver and pancreas.

Tributyltin chloride (TBT) is an environmental contaminant used in antifouling paints of boats. Endocrine disruptor effects of TBT are well established in animal models. However, the adverse effects on metabolism are less well understood. The toxicity of TBT in the white adipose tissue (WAT), liver and pancreas of female rats were assessed. Animals were divided into control and TBT (0.1 µg/kg/day) groups. TBT induced an increase in the body weight of the rats by the 15th day of oral exposure. The weight gain was associated with high parametrial (PR) and retroperitoneal (RP) WAT weights. TBT-treatment increased the adiposity, inflammation and expression of ERα and PPARγ proteins in both RP and PR WAT. In 3T3-L1 cells, estrogen treatment reduced lipid droplets accumulation, however increased the ERα protein expression. In contrast, TBT-treatment increased the lipid accumulation and reduced the ERα expression. WAT metabolic changes led to hepatic inflammation, lipid accumulation, increase of PPARγ and reduction of ERα protein expression. Accordingly, there were increases in the glucose tolerance and insulin sensitivity tests with increases in the number of pancreatic islets and insulin levels. These findings suggest that TBT leads to adiposity in WAT specifically, impairing the metabolic functions of the liver and pancreas.

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.

Endogenous female sex hormones delay the development of renal dysfunction in apolipoprotein E-deficient mice.

BACKGROUND: Hypercholesterolemia is a well-established risk factor for the development of kidney injury. Considering that female sex hormones may play a preventative role in both cardiovascular and renal diseases, the aim of the present study was to evaluate the effects of female sex hormones on hypercholesterolemia-induced renal dysfunction. METHODS: Apolipoprotein E-deficient (ApoE) and C57 control female mice underwent an ovariectomy (OVX) or sham surgery and after 2 months, creatinine clearance, uremia and proteinuria were determined. Renal oxidative stress and lipid deposition were also quantified. Values are presented as mean ± SEM. Statistical analyses were performed using Two-way ANOVA followed by Tukey's post hoc test. RESULTS: Creatinine clearance (µL/min) was similar between C57 (171 ± 17) and ApoE (140 ± 26) mice underwent sham surgery. OVX resulted in a reduced glomerular filtration rate in both C57 (112 ± 8, ~ - 35%, p < 0.05) and ApoE (61 ± 10, ~ - 56%, p < 0.05) animals. Plasma levels of urea (mg/dL) were higher in both ApoE groups (Sham: 73 ± 7; OVX: 73 ± 8, p < 0.05) when compared to C57 animals (Sham: 49 ± 3; OVX: 60 ± 4), with no changes among ovariectomized groups. Proteinuria levels (mg/24 h) were similar between C57 (Sham: 25.1 ± 5.7; OVX: 33.7 ± 4.7) and ApoE sham animals (26.4 ± 3.5), however, 24-h urine protein excretion was augmented in ApoE OVX animals (49.6 ± 5.8, p < 0.05). Histological kidney analysis demonstrated that the absence of female sex hormones resulted in increased oxidative stress, which was more severe in ApoE mice (C57 Sham: 9.2 ± 0.4; C57 OVX: 22.9 ± 1.0; ApoE Sham: 13.9 ± 0.7; ApoE OVX: 34.0 ± 1.4 au x 103, p < 0.05). As expected, ApoE mice presented higher lipid deposition, which was not affected by OVX (C57 Sham: 0 ± 0; C57 OVX: 0 ± 0; ApoE Sham: 6.8 ± 1.6; ApoE OVX: 5.2 ± 0.8% x 10-2, p < 0.05). Ovariectomy resulted in a similar reduction in ER-α protein expression in the renal cortex (C57: 0.78 ± 0.04; ApoE: 0.81 ± 0.04 au, p < 0.05) when compared to sham animals (C57:1.00 ± 0.04; ApoE: 1.03 ± 0.03 au). CONCLUSION: Taken together these data indicate that female sex hormones may delay hypercholesterolemia-induced renal dysfunction and emphasizes the importance of plasma cholesterol control in post-menopausal women.

Inhibition of phosphodiesterase 5 restores endothelial function in renovascular hypertension.

BACKGROUND: The clipping of an artery supplying one of the two kidneys (2K1C) activates the renin-angiotensin (Ang) system (RAS), resulting in hypertension and endothelial dysfunction. Recently, we demonstrated the intrarenal beneficial effects of sildenafil on the high levels of Ang II and reactive oxygen species (ROS) and on high blood pressure (BP) in 2K1C mice. Thus, in the present study, we tested the hypothesis that sildenafil improves endothelial function in hypertensive 2K1C mice by improving the NO/ROS balance. METHODS: 2K1C hypertension was induced in C57BL/6 mice. Two weeks later, they were treated with sildenafil (40 mg/kg/day, via oral) or vehicle for 2 weeks and compared with sham mice. At the end of the treatment, the levels of plasma and intrarenal Ang peptides were measured. Endothelial function and ROS production were assessed in mesenteric arterial bed (MAB). RESULTS: The 2K1C mice exhibited normal plasma levels of Ang I, II and 1-7, whereas the intrarenal Ang I and II were increased (~35% and ~140%) compared with the Sham mice. Sildenafil normalized the intrarenal Ang I and II and increased the plasma (~45%) and intrarenal (+15%) Ang 1-7. The 2K1C mice exhibited endothelial dysfunction, primarily due to increased ROS and decreased NO productions by endothelial cells, which were ameliorated by treatment with sildenafil. CONCLUSION: These data suggest that the effects of sildenafil on endothelial dysfunction in 2K1C mice may be due to interaction with RAS and restoring NO/ROS balance in the endothelial cells from MAB. Thus, sildenafil is a promising candidate drug for the treatment of hypertension accompanied by endothelial dysfunction and kidney disease.

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.