Potentiation of anti-angiogenic eNOS-siRNA transfection by ultrasound-mediated microbubble destruction in ex vivo rat aortic rings.

Nitric oxide (NO) regulates vascular homeostasis and plays a key role in revascularization and angiogenesis. The endothelial nitric oxide synthase (eNOS) enzyme catalyzes NO production in endothelial cells. Overexpression of the eNOS gene has been implicated in pathologies with dysfunctional angiogenic processes, such as cancer. Therefore, modulating eNOS gene expression using small interfering RNAs (siRNAs) represents a viable strategy for antitumor therapy. siRNAs are highly specific to the target gene, thus reducing off-target effects. Given the widespread distribution of endothelium and the crucial physiological role of eNOS, localized delivery of nucleic acid to the affected area is essential. Therefore, the development of an efficient eNOS-siRNA delivery carrier capable of controlled release is imperative for targeting specific vascular regions, particularly those associated with tumor vascular growth. Thus, this study aims to utilize ultrasound-mediated microbubble destruction (UMMD) technology with cationic microbubbles loaded with eNOS-siRNA to enhance transfection efficiency and improve siRNA delivery, thereby preventing sprouting angiogenesis. The efficiency of eNOS-siRNA transfection facilitated by UMMD was assessed using bEnd.3 cells. Synthesis of nitric oxide and eNOS protein expression were also evaluated. The silencing of eNOS gene in a model of angiogenesis was assayed using the rat aortic ring assay. The results showed that from 6 to 24 h, the transfection of fluorescent siRNA with UMMD was twice as high as that of lipofection. Moreover, transfection of eNOS-siRNA with UMMD enhanced the knockdown level (65.40 ± 4.50%) compared to lipofectamine (40 ± 1.70%). Silencing of eNOS gene with UMMD required less amount of eNOS-siRNA (42 ng) to decrease the level of eNOS protein expression (52.30 ± 0.08%) to the same extent as 79 ng of eNOS-siRNA using lipofectamine (56.30 ± 0.10%). NO production assisted by UMMD was reduced by 81% compared to 67% reduction transfecting with lipofectamine. This diminished NO production led to higher attenuation of aortic ring outgrowth. Three-fold reduction compared to lipofectamine transfection. In conclusion, we propose the combination of eNOS-siRNA and UMMD as an efficient, safe, non-viral nucleic acid transfection strategy for inhibition of tumor progression.

Cellular shortening and calcium dynamics are improved by noisy stimulus in a model of cardiomyopathy.

Noise is present in cell biology. The capability of cells to respond to noisy environment have become essential. This study aimed to investigate whether noise can enhance the contractile response and Ca2+ handling in cardiomyocytes from a cardiomyopathy model. Experiments were conducted in an experimental setup with Gaussian white noise, frequency, and amplitude control to stimulate myocytes. Cell shortening, maximal shortening velocity, time to peak shortening, and time to half relaxation variables were recorded to cell shortening. Ca2+ transient amplitude and raise rate variables were registered to measure Ca2+ transients. Our results for cell shortening, Ca2+ transient amplitude, and raise rate suggest that cell response improve when myocytes are noise stimulated. Also, cell shortening, maximal shortening velocity, Ca2+ transient amplitude, and raise improves in control cells. Altogether, these findings suggest novel characteristics in how cells improve their response in a noisy environment.

Caveolin Delivered by Ultrasound-Mediated Microbubble Destruction Prevents Endothelial Cell Proliferation.

Introduction: The nitric oxide synthase (eNOS) is an important regulator of vascular homeostasis. eNOS is modulated by intracellular mechanisms that include protein-protein interaction with Caveolin-1 (Cav). Cav binds to and impairs eNOS activation reducing vascular permeability and angiogenesis. Blocking of eNOS by Cav has been proposed as therapeutic antiangiogenic approach. However, the efficient and controlled delivery of the peptide requires to be solved. Methods: The effect of antennapedia (AP)-Cav loaded into microbubbles (MBs) and delivered by ultrasound-mediated microbubble destruction (UMMD) into brain endothelial cells (bEnd.3 cells) was evaluated on NO production using DAF2-DA, cell migration assessed by the wound healing assay, cell proliferation with BrdU, and ex-vivo angiogenesis in rat aortic rings. Results: An enhanced inhibitory effect of AP-Cav was observed on cells treated with UMMD. MBs and ultrasound disruption delivery of AP-Cav increased acetylcholine-induced NO release, wound healing, cell proliferation, and angiogenesis inhibition on bEnd.3 cells, compared to free AP-Cav administration. Conclusion: We demonstrated that the delivery of Cav via AP-Cav-loaded MBs and UMMD may be an administration method for Cav that would increase its therapeutic potential by enhancing efficacy and cellular specificity.

Computer-assisted image analysis of agonist-mediated microvascular constriction response in mouse cremaster muscle.

In this work, we implemented an automated method using a correlation coefficient to select a time interval with a minimum movement or rest interval, together with analysis of variance for measurement of blood vessel diameter in the cremaster muscle. Video images binarization using analysis of variance resulted in an enhanced and a clearly defined vessel wall. Histamine (1 mM) induced a marked reduction in vascular diameter (vasoconstriction) in the cremaster muscle from mice fed with standard (SD) and high fat diet (HFD). However, the effect of histamine was reduced in HFD mice compared to SD mice. Thus, the change in vascular diameter was 87.14% ± 7.44% and 52.63% ± 16.27% in SD and HFD mice, respectively. In conclusion, determination of a rest interval with minimal movement and the use of analysis of variance resulted useful to evaluate vascular diameter in small arteries. We suggest this method to streamline experiments facilitating cardiovascular research.

Use of Nitric Oxide Donor-Loaded Microbubble Destruction by Ultrasound in Thrombus Treatment.

In the presence of a vascular thrombus, the recovery of blood flow and vascular recanalization are very important to prevent tissue damage. An alternative procedure to thrombolysis is required for patients who are unable to receive surgery or thrombolytic drugs due to other physical conditions. Recently, the performance of thrombolysis combined with microbubbles has become an attractive and effective therapeutic procedure. Indeed, in a recent study, we demonstrated that, upon exposure to ultrasound, liposomes loaded with nitric oxide release agonists conjugated to microbubbles; therefore, there is potential to release the agonist in a controlled manner into specific tissues. This means that the effect of the agonist is potentiated, decreasing interactions with other tissues, and reducing the dose required to induce nitric-oxide-dependent vasodilation. In the present study, we hypothesized that a liposome microbubble delivery system can be used as a hydrophilic agonist carrier for the nitric oxide donor spermine NONOate, to elicit femoral vasodilation and clot degradation. Therefore, we used spermine-NONOate-loaded microbubbles to evaluate the effect of ultrasound-mediated microbubble disruption (UMMD) on thromboembolic femoral artery recanalization. We prepared spermine NONOate-loaded microbubbles and tested their effect on ex vivo preparations, hypothesizing that ultrasound-induced microbubble disruption is associated with the vasorelaxation of aortic rings. Thrombolysis was demonstrated in aorta blood-flow recovery after disruption by spermine NONOate-loaded microbubbles via ultrasound application in the region where the thrombus is located. Our study provides an option for the clinical translation of NO donors to therapeutic applications.

Thromboxane-dependent coronary vasoconstriction in obese mice: Role of peroxynitrite.

Obesity leads to chronic oxidative stress promoting the development of cardiovascular diseases including coronary artery disease and endothelial dysfunction. Increased reactive oxygen species production associated with obesity might lead to endothelial dysfunction through cyclooxygenase (COX) pathway. We evaluated arachidonic acid (AA)-dependent coronary vascular responses and explored COX metabolism in obese C57BL/6 mice. In response to arachidonic acid (AA), isolated hearts from obese mice showed increased vasoconstriction compared with control mice. Released thromboxane (TX) A2 during AA-induced vasoconstriction phase was increased in heart perfusates from obese mice. Indomethacin and 1-benzylimidazole, both reduced vasoconstriction response in control and obese mice. Vasoconstriction response to TXA2 mimetic analog U46619 was 2.7 higher in obese mice. Obesity increased COX-2, TXS and TX receptor protein expression as well as oxidative stress evaluated by nitrotyrosine and peroxynitrite levels, compared with control mice. Obese mice treated with FeTMPyP, a peroxynitrite scavenger, reversed all these parameters to control levels. These data suggest that alterations in COX pathway may be associated with increased generation of free radicals, including peroxynitrite, that result from the oxidative stress observed in obesity.

Release of vascular agonists from liposome-microbubble conjugate by ultrasound-mediated microbubble destruction: effect on vascular function.

The endothelium is a single cell layer of the vessel wall and a key regulator of blood flow in vascular beds. Local and systemic pathologies have been associated with alterations in endothelial function. However, targeting the endothelium with vasoconstrictor or vasodilator drugs is often accompanied by systemic effects. Here, we evaluated a liposome-microbubble delivery system as a vascular hydrophilic agonist carrier. Phenylephrine (Phe) or acetylcholine (Ach)-loaded liposomes were conjugated to microbubbles. The drug release was triggered by ultrasound (US), and the vascular response was assessed in rat aortic rings using an isolated organ chamber. Aortic rings incubated with Phe-liposome-microbubble conjugate, exposed to US showed a marked contractile response (0.79 ± 0.04 g) compared to empty liposomes conjugated to microbubbles, aortic rings exposed only to US, and Phe-liposome-microbubble conjugate without US exposure that elicited a minimal or no response. Expressed as %, contractile responses were 85.24 ± 4.31% and 12.62 ± 3.23% for Phe-Chol-liposome-microbubble conjugate and empty Chol-liposome-microbubble conjugate exposed to US, respectively. Addition of 1 × 10-5 M Ach to pre-contracted aortic rings decreased the contraction response from 1 to 0.21 g. The addition of Ach-liposome conjugate and exposure to US decreased the contraction response to 0.32 g. Additionally, the ED50 values for Phe and Ach released by US from liposome-microbubble conjugates were 3.6 × 10-8 M ± 2.8 × 10-9 M for Phe and 2.0 × 10-8 M ± 1.8 × 10-9 M. In conclusion, we evaluated a hybrid delivery system that consisted of loaded liposomes conjugated to microbubbles to deliver and release vascular agonists using UMMD.

Localized Delivery of Caveolin-1 Peptide Assisted by Ultrasound-Mediated Microbubble Destruction Potentiates the Inhibition of Nitric Oxide-Dependent Vasodilation Response.

In the endothelium, nitric oxide synthase (eNOS) is the enzyme that generates nitric oxide, a key molecule involved in a variety of biological functions and cancer-related events. Therefore, selective inhibition of eNOS represents an attractive therapeutic approach for NO-related diseases and anticancer therapy. Ultrasound-mediated microbubble destruction (UMMD) conjugated with cell-permeable peptides has been investigated as a drug delivery system for effective delivery of anticancer molecules. We investigated the feasibility of loading antennapedia-caveolin-1 peptide (AP-Cav), a specific eNOS inhibitor, onto microbubbles to be delivered by UMMD in rat aortic endothelium. AP-Cav-loaded microbubbles (AP-Cav-MBs) and US parameters were characterized. Aortas were treated with UMMD for 30 s with 1.3 × 108 MBs/mL AP-Cav (8 µM)-MBs at 100-Hz pulse repetition frequency, 0.5-MPa acoustic pressure, 0.5 mechanical index and 10% duty cycle. NO-dependent vascular responses were assessed using an isolated organ system, 21 h post-treatment. Maximal relaxation response was inhibited 61.8% ± 1.6% in aortas treated with UMMD-AP-Cav-MBs, while in aortas treated with previously disrupted AP-Cav-MBs and then US, the inhibition was 31.6% ± 1.6%. The vascular contractile response was not affected. The impact of UMMD was evaluated in aortas treated with free AP-Cav; 30 µM of free AP-Cav was necessary to reach an inhibition response similar to that obtained with UMMD-AP-Cav-MBs. In conclusion, UMMD enhances the delivery and potentiates the effect of AP-Cav in the endothelial layer of rat aorta segments.

Functional Activity and Endothelial-Lining Integrity of Ex Vivo Arteries Exposed to Ultrasound-Mediated Microbubble Destruction.

Ultrasound-mediated microbubble destruction (UMMD) is a promising strategy to improve local drug delivery in specific tissues. However, acoustic cavitation can lead to harmful bioeffects in endothelial cells. We investigated the side effects of UMMD treatment on vascular function (contraction and relaxation) and endothelium integrity of ex vivo Wistar rat arteries. We used an isolated organ system to evaluate vascular responses and confocal microscopy to quantify the integrity and viability of endothelial cells. The arteries were exposed for 1-3 min to ultrasound at a 100 Hz pulse-repetition frequency, 0.5 MPa acoustic pressure, 50% duty cycle and 1%-5% v/v microbubbles. The vascular contractile response was not affected. The acetylcholine-dependent maximal relaxation response was reduced from 78% (control) to 60% after 3 min of ultrasound exposure. In arteries treated simultaneously with 1 min of ultrasound exposure and 1%, 2%, 3% or 5% microbubble concentration, vascular relaxation was reduced by 19%, 58%, 80% or 93%, respectively, compared with the control arteries. Fluorescent labeling revealed that apoptotic death, detachment of endothelial cells and reduced nitric oxide synthase phosphorylation are involved in relaxation impairment. We demonstrated that UMMD can be a safe technology if the correct ultrasound and microbubble parameters are applied. Furthermore, we found that tissue-function evaluation combined with cellular analysis can be useful to study ultrasound-microbubble-tissue interactions in the optimization of targeted endothelial drug delivery.

Role of sodium/glucose cotransporter inhibition on a rat model of angiotensin II-dependent kidney damage.

BACKGROUND: Renal proximal tubular sodium and glucose reabsorption are regulated by the sodium-glucose cotransporter (SGLT2). Changes in this transporter can play a role in hyperglycaemia and reactive oxygen species (ROS) production. We demonstrated increased glucose absorption in proximal tubule membrane vesicles and increased expression of SGLT2 in hypertensive rats. Here we investigated Angiotensin II (Ang II) -dependent SGLT2 expression induction and the role of SGLT2 induction in the development of Ang II-dependent kidney damage. The aim of this study was to determine whether SGLT2 induction by Ang II is associated with Ang II-dependent kidney damage. We propose the following objectives a) to demonstrate that Ang II induces SGLT2 expression and b) to demonstrate that prevention of SGLT2 expression and activity prevent Ang II-induced kidney damage. METHODS: We used chronic Ang II infusion as a model of kidney damage in male Wistar rats and evaluated systolic blood pressure by telemetric methods. SGLT2 mRNA and protein expression were evaluated by PCR and immunoblotting. SGLT2 activity was evaluated in brush border membrane vesicles by measuring glucose uptake. ROS production was measured by confocal microscopy. The glomerular filtration rate (GFR) was evaluated by the inulin excretion method, and urinary protein excretion was evaluated by the Bradford method. Biological parameter evaluations were performed, after two weeks of infusion of Ang II. We compared the effects of Angiotensin II (AT1) receptor blockade by Losartan and SGLT2 inhibition by Empagliflozin both as monotherapy treatments and in combination on the development of kidney damage. RESULTS: Chronic Ang II infusion led to a blood pressure elevation and increased SGLT2 mRNA expression and activity as well as kidney damage, as reflected by increased ROS production, decreased GFR and increased urinary protein excretion. AT1 receptor blockade prevented all these changes. By contrast, SGLT2 inhibition did not affect blood pressure and had a small effect on kidney damage. However, the combination of both drugs resulted in the potentiation of the effects observed by AT1 receptor blockade alone. CONCLUSIONS: We suggest that Ang II-dependent increased SGLT2 induction is one mechanism by which Ang II induces kidney damage.

Abnormal synchronization patterns in the electrical stimulation-contractile response coupling decrease with noise.

Synchronization theory predicts that if an oscillator interacts with a rhythmical external force, then it should react to a rhythmical force by adjusting its frequency. Furthermore, noise is present in nature, and it affects the nervous and cardiovascular systems. In this paper, we analyze the heart as an oscillator, where noisy periodic electrical stimulation can be regarded as an external forcing. This study aimed to investigate, from an experimental point of view, whether noise can induce synchronization of higher order in the mechanical heart response. A Langendorff heart preparation was used to obtain two variables of the mechanical response, intensity of contractile force and heart rate. The experiments show frequency locking in the electrical stimulation-contractile response coupling with and without noise induced. The role of noise in the response of effector organs invites further investigation.

Comparative effects of nitric oxide dependent and independent vasodilation on impaired hindlimb revascularization in eNOS-/- mice.

Ischemia due to vascular occlusion induces vasodilation as an initial response, followed by arteriogenesis or angiogenesis. Vasodilation through nitric oxide (NO) independent and dependent mechanisms may be sufficient to restore the altered neovascularization in pathological situations where the NO is altered. Using a posterior limb claudication model to evaluate ischemia-induced revascularization in eNOS-/- mice, we compared the effects of sodium nitrite, a NO-dependent vasodilator, and prazocin, an alpha-adrenergic blocker and NO-independent vasodilator, on hindlimb revascularization. We evaluated the blood flow of the hindlimbs, NO and nitrites metabolites, the expression of tissue endothelial cell markers and proangiogenic factors, as well as the gait locomotion. Our results suggest that the use of a peripheral vasodilator can substitute the initial absence of NO as an endogenous vasodilator. However, final resolution of the ischemic process requires a NO-mediated pathway, which through changes in vascular hemodynamics, promotes the generation of angiogenic messengers facilitating the functional recovery of the damaged limb.

An Affordable and Portable Thermocycler for Real-Time PCR Made of 3D-Printed Parts and Off-the-Shelf Electronics.

The polymerase chain reaction (PCR) is a sought-after nucleic acid amplification technique used in the detection of several diseases. However, one of the main limitations of this and other nucleic acid amplification assays is the complexity, size, maintenance, and cost of their operational instrumentation. This limits the use of PCR applications in settings that cannot afford the instruments but that may have access to basic electrical, electronic, and optical components and the expertise to build them. To provide a more accessible platform, we developed a low-cost, palm-size, and portable instrument to perform real-time PCR (qPCR). The thermocycler leverages a copper-sheathed power resistor and a computer fan, in tandem with basic electronic components controlled from a single-board computer. The instrument incorporates a 3D-printed chassis and a custom-made fluorescence optical setup based on a CMOS camera and a blue LED. Results are displayed in real-time on a tablet. We also fabricated simple acrylic microdevices consisting of four wells (2 µL in volume each) where PCR reactions take place. To test our instrument, we performed qPCR on a series of cDNA dilutions spanning 4 orders of magnitude, achieving similar limits of detection as those achieved by a benchtop thermocycler. We envision our instrument being utilized to enable routine monitoring and diagnosis of certain diseases in low-resource areas.

Androgens Mediate ß-adrenergic Vasorelaxation Impairment Using Adenylyl Cyclase.

Cardiovascular disease development has been associated with sex differences, suggesting that sex hormones are implicated in vascular function and development of hypertension. Vascular tone comparison at different stages of rat growth represents a good model to study testosterone-related vascular response. We explored the role of testosterone in modulation of age-dependent impaired ß-adrenergic vasodilation. The 3-week-old male Sprague-Dawley rats were sorted in 3-week-old rats without any manipulation and 3-week-old rats treated with testosterone. The 9-week-old rats were randomly grouped into 9-week-old rats without any manipulation (sham), 9-week-old rats that underwent gonadectomy (9-week-old castrated), and 9-week-old castrated treated with testosterone replacement therapy (9-week-old castrated + testosterone). Vascular relaxation was evaluated in aortic rings. ß-adrenergic receptor protein expression, cyclic adenosine monophosphate production, testosterone levels, and adenylyl cyclase (AC) gene expression were assessed. Testosterone levels were low in 3-week-old and 9-week-old castrated rats compared with 9-week-old sham rats. Testosterone replacement raised these levels in 3-week-old and 9-week-old castrated rats similar to those of 9-week-old sham rats. SQ22536, the AC inhibitor, prevented isoproterenol-induced relaxation in aortic rings from 3-week-old and 9-week-old castrated rats. The ß-adrenergic receptor protein expression was similar in all experimental groups. AC mRNA and protein expression and cyclic adenosine monophosphate levels were elevated in 3-week-old and 9-week-old castrated rats compared with 3-week-old + testosterone, 9-week-old sham, and 9-week-old castrated + testosterone rats. In conclusion, we demonstrated that age maturation was associated with vascular relaxation impairment. Variations in testosterone levels and reduced AC expression may be responsible for this altered vascular function.

Noise enhanced the electrical stimulation-contractile response coupling in isolated mouse heart.

BACKGROUND: Stochastic resonance is a phenomenon that allows a system to improve its capability to detect stimulus when a limited amount of noise is added to the stimuli. It has experimentally been shown that noise enhances the homeostatic function of the blood pressure regulatory system. This study aimed to investigate whether the noise can enhance the contractile response in the whole heart. METHODS: Experiments were conducted in isolated mouse hearts (0.040kg, n=8), a Langendorff heart preparation is used to obtain two variables of the contractile response contraction force and heart rate. The contractile response due to an electrical stimulation perturbed with Gaussian noise was recorded. RESULTS: The results show that the intensity of noise induced in the electrical stimuli has an effect on the electrical stimulation-contractile response coupling. With 10% noise induced, the bandwidth where the synchronization effect is presented was increased from (7-11Hz) to (6-12Hz), and the irregular dynamic threshold was changed to 13Hz. CONCLUSIONS: We find that the noise increases the synchronization bandwidth in the electrical stimulation-contractile response coupling. We have experimentally demonstrated the stochastic resonance in isolated mouse heart.

Combined effects of flow-induced shear stress and electromagnetic field on neural differentiation of mesenchymal stem cells.

Differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into neural phenotype has been induced by either flow-induced shear stress (FSS) or electromagnetic fields (EMF). However, procedures are still expensive and time consuming. In the present work, induction for 1 h with the combination of both forces showed the presence of the neural precursor nestin as early as 9 h in culture after treatment and this result lasted for the following 6 d. In conclusion, the use of a combination of FSS and EMF for a short-time renders in neurite-like cells, although further investigation is required to analyze cell functionality.

Oxidative Stress-Dependent Coronary Endothelial Dysfunction in Obese Mice.

Obesity is involved in several cardiovascular diseases including coronary artery disease and endothelial dysfunction. Endothelial Endothelium vasodilator and vasoconstrictor agonists play a key role in regulation of vascular tone. In this study, we evaluated coronary vascular response in an 8 weeks diet-induced obese C57BL/6 mice model. Coronary perfusion pressure in response to acetylcholine in isolated hearts from obese mice showed increased vasoconstriction and reduced vasodilation responses compared with control mice. Vascular nitric oxide assessed in situ with DAF-2 DA showed diminished levels in coronary arteries from obese mice in both basal and acetylcholine-stimulated conditions. Also, released prostacyclin was decreased in heart perfusates from obese mice, along with plasma tetrahydrobiopterin level and endothelium nitric oxide synthase dimer/monomer ratio. Obesity increased thromboxane A2 synthesis and oxidative stress evaluated by superoxide and peroxynitrite levels, compared with control mice. Obese mice treated with apocynin, a NADPH oxidase inhibitor, reversed all parameters to normal levels. These results suggest that after 8 weeks on a high-fat diet, the increase in oxidative stress lead to imbalance in vasoactive substances and consequently to endothelial dysfunction in coronary arteries.

Antioxidative diet supplementation reverses high-fat diet-induced increases of cardiovascular risk factors in mice.

Obesity is a worldwide epidemic that is characterized not only by excessive fat deposition but also by systemic microinflammation, high oxidative stress, and increased cardiovascular risk factors. While diets enriched in natural antioxidants showed beneficial effects on oxidative stress, blood pressure, and serum lipid composition, diet supplementation with synthetic antioxidants showed contradictive results. Thus, we tested in C57Bl/6 mice whether a daily dosage of an antioxidative mixture consisting of vitamin C, vitamin E, L-arginine, eicosapentaenoic acid, and docosahexaenoic acid (corabion) would affect cardiovascular risk factors associated with obesity. Obese mice showed increased serum triglyceride and glucose levels and hypertension after eight weeks of being fed a high-fat diet (HFD). Importantly, corabion ameliorated all of these symptoms significantly. Oxidative stress and early signs of systemic microinflammation already developed after two weeks of high-fat diet and were significantly reduced by daily doses of corabion. Of note, the beneficial effects of corabion could not be observed when applying its single antioxidative components suggesting that a combination of various nutrients is required to counteract HFD-induced cardiovascular risk factors. Thus, daily consumption of corabion may be beneficial for the management of obesity-related cardiovascular complications.

Early stage of obesity potentiates nitric oxide reduction during the development of renal failure.

BACKGROUND: Obesity is a serious health problem associated with the pathogenesis of various metabolic diseases. Nitric Oxide (NO) plays an important role in kidney function and altered NO levels have been associated with the pathogenesis of obesity. Therefore, we aimed to study whether an early stage of obesity contributes with progression of renal failure through further NO impairment. METHODS: Male C57BL/6 mice were fed with a high-fat diet (HFD) or a normal diet (ND) during 2 weeks. All mice underwent either sham surgery (sham) or 5/6 nephrectomy (Np). One group of HFD Np mice was treated with antioxidants plus L-arginine. Kidney damage parameters were assessed and eNOS metabolism was evaluated. RESULTS: Mice on a HFD increased body weight, eNOS protein and mRNA expression, and radical oxygen species (ROS). Urine nitrites excretion, urine volume, and plasma BH4 were decreased. In HFD mice, 5/6 Np further increased BH2 and urine protein concentration, ROS levels, and eNOS mRNA expression. The decrease in BH4 plasma levels and urine nitrites excretion was accentuated. NO synthesis stimulation with the antioxidants + L-arginine treatment prevented all these changes. CONCLUSIONS: The early changes in NO metabolism are associated with an early stage of obesity. This effect on NO potentiates kidney damage development.

Cyclooxygenase-2 and kidney failure.

Cyclooxygenase (COX)-dependent prostaglandins are necessary for normal kidney function. These prostaglandins are associated with inflammation, maintenance of sodium and water homeostasis, control of renin release, renal vasodilation, vasoconstriction attenuation, and prenatal renal development. COX-2 expression is regulated by the renin-angiotensin system, glucocorticoids or mineralcorticoids, and aldosterone, supporting a role for COX-2 in kidney function. Indeed, COX-2 mRNA and protein levels as well as enzyme activity are increased, along with PGE2, during kidney failure. In addition, changes in COX-2 expression are associated with increased blood pressure, urinary volume, sodium and protein and decreased urinary osmolarity. Intrarenal mechanisms such as angiotensin II (Ang II) production, increased sodium delivery, glomerular hypertension, and renal tubular inflammation have been suggested to be responsible for the increase in COX-2 expression. Although, specific COX-2 pharmacological inhibition has been related to the prevention of kidney damage, clinical studies have reported that COX-2 inhibition may cause side effects such as edema or a modest elevation in blood pressure and could possibly interfere with antihypertensive drugs and increase the risk of cardiovascular complications. Thus, administration of COX-2 inhibitors requires caution, especially in the presence of underlying cardiovascular disease.