Leptin Induces Oxidative Stress Through Activation of NADPH Oxidase in Renal Tubular Cells: Antioxidant Effect of L-Carnitine.

Leptin is a protein involved in the regulation of food intake and in the immune and inflammatory responses, among other functions. Evidences demonstrate that obesity is directly associated with high levels of leptin, suggesting that leptin may directly link obesity with the elevated cardiovascular and renal risk associated with increased body weight. Adverse effects of leptin include oxidative stress mediated by activation of NADPH oxidase. The aim of this study was to evaluate the effect of L-carnitine (LC) in rat renal epithelial cells (NRK-52E) exposed to leptin in order to generate a state of oxidative stress characteristic of obesity. Leptin increased superoxide anion (O2 (•) -) generation from NADPH oxidase (via PI3 K/Akt pathway), NOX2 expression and nitrotyrosine levels. On the other hand, NOX4 expression and hydrogen peroxide (H2 O2 ) levels diminished after leptin treatment. Furthermore, the expression of antioxidant enzymes, catalase, and superoxide dismutase, was altered by leptin, and an increase in the mRNA expression of pro-inflammatory factors was also found in leptin-treated cells. LC restored all changes induced by leptin to those levels found in untreated cells. In conclusion, stimulation of NRK-52E cells with leptin induced a state of oxidative stress and inflammation that could be reversed by preincubation with LC. Interestingly, LC induced an upregulation of NOX4 and restored the release of its product, hydrogen peroxide, which suggests a protective role of NOX4 against leptin-induced renal damage. J. Cell. Biochem. 117: 2281-2288, 2016. © 2016 Wiley Periodicals, Inc.

The renoprotective effect of L-carnitine in hypertensive rats is mediated by modulation of oxidative stress-related gene expression.

PURPOSE: Arterial hypertension is associated with a high production of reactive oxygen species and a decrease in the antioxidant defense systems. Based on the lack of toxicity of L-carnitine (LC) and previous studies reporting beneficial effects of this compound in experimental models of hypertension, the aim of this work was to test the hypothesis that LC might protect the kidney against hypertension-induced oxidative damage, as well as to investigate the mechanisms involved in this effect. To this end, specific activities and protein/mRNA expression of the antioxidant enzymes (glutathione peroxidase, glutathione reductase, and superoxide dismutase), and those of NADPH oxidase (the main responsible for superoxide anion production in renal tissue) have been measured in renal cortex homogenates from NG-nitro-L-arginine methyl ester (L-NAME)-treated rats and control normotensive rats. In addition, components of the renin-angiotensin system (RAS) and redox-sensitive transcription factors (NF-κB, Nrf2, and PPARα) have also been evaluated. METHODS: Male Wistar rats aged 6-8 weeks were divided into four groups of six animals each: (1) control, normotensive Wistar rats (with free access to tap water); (2) Wistar rats subjected to treatment with 25 mg of L-NAME/kg body weight/day dissolved in the drinking water, in order to develop L-NAME-induced hypertension; (3) Wistar rats subjected to treatment with 400 mg of LC/kg body weight/day (also dissolved in the drinking water); and (4) L-NAME-treated rats subjected to simultaneous treatment with LC at the indicated doses. RESULTS: The beneficial effect of LC supplementation on oxidative damage in the renal cortex of hypertensive rats reversed hypertension-associated renal function damage and produced an upregulation of both antioxidant enzymes and eNOS, and with a downregulation of both NADPH oxidase and RAS components. LC improves the oxidative stress response through a specific modulation of NF-κB, Nrf2, and PPARα transcription factors. Thus, the low production of superoxide anions, subsequent to NADPH oxidase inhibition, might act by increasing the expression of Nrf2 and PPARα and by decreasing that of NF-κB, which, in turn, would enhance the antioxidant defense systems. CONCLUSIONS: Our results might support the use of LC to prevent hypertension-induced renal damage.

Biostimulant Capacity of an Enzymatic Extract From Rice Bran Against Ozone-Induced Damage in Capsicum annum.

Ozone is a destructive pollutant, damaging crops, and decreasing crop yield. Therefore, there is great interest in finding strategies to alleviate ozone-induced crop losses. In plants, ozone enters leaves through the stomata and is immediately degraded into reactive oxygen species (ROS), producing ROS stress in plants. ROS stress can be controlled by ROS-scavenging systems that include enzymatic or non-enzymatic mechanisms. Our research group has developed a product from rice bran, a by-product of rice milling which has bioactive molecules that act as an antioxidant compound. This product is a water-soluble rice bran enzymatic extract (RBEE) which preserves all the properties and improves the solubility of proteins and the antioxidant components of rice bran. In previous works, the beneficial properties of RBEE have been demonstrated in animals. However, to date, RBEE has not been used as a protective agent against oxidative damage in agricultural fields. The main goal of this study was to investigate the ability of RBEE to be used as a biostimulant by preventing oxidative damage in plants, after ozone exposure. To perform this investigation, pepper plants (Capsicum annuum) exposed to ozone were treated with RBEE. RBEE protected the ozone-induced damage, as revealed by net photosynthetic rate and the content of photosynthetic pigments. RBEE also decreased the induction of antioxidant enzyme activities in leaves (catalase, superoxide dismutase, and ascorbate peroxidase) due to ozone exposure. ROS generation is a common consequence of diverse cellular traumas that also activate the mitogen-activated protein kinase (MAPK) cascade. Thus, it is known that the ozone damages are triggered by the MAPK cascade. To examine the involvement of the MAPK cascade in the ozone damage CaMPK6-1, CaMPK6-2, and CaMKK5 genes were analyzed by qRT-PCR. The results showed the involvement of the MAPK pathway in both, not only in ozone damage but especially in its protection by RBEE. Taken together, these results support that RBEE protects plants against ozone exposure and its use as a new biostimulant could be proposed.

Protease technology for obtaining a soy pulp extract enriched in bioactive compounds: isoflavones and peptides.

This work presents a new bioprocess process for the extraction of bioactive components from soy pulp by-product (okara) using an enzymatic technology that was compared to a conventional water extraction. Okara is rich in fiber, fat, protein, and bioactive compounds such as isoflavones but its low solubility hampers the use in food and fertilizer industry. After the enzymatic attack with endoproteases half of the original insoluble proteins were converted into soluble peptides. Linked to this process occured the solubilization of isoflavones trapped in the insoluble protein matrix. We were able to extract up to 62.5% of the total isoflavones content, specially aglycones, the more bioactive isoflavone forms, whose values rose 9.12 times. This was probably due to the increased solubilization and interconversion from the original isoflavones. In conclusion, our process resulted in the formulation of a new functional product rich in aglycones and bioactive peptides with higher antioxidant potency than the original source. Therefore, we propose that the enzymatic extraction of okara bioactive compounds is an advantageous tool to replace conventional extraction.

L-carnitine attenuates oxidative stress in hypertensive rats.

The present study aimed to investigate whether l-carnitine (LC) protects the vascular endothelium and tissues against oxidative damage in hypertension. Antioxidant enzyme activities, glutathione and lipid peroxidation were measured in the liver and heart of spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. Nitrite and nitrate levels and total antioxidant status (TAS) were evaluated in plasma, and the expression of endothelial nitric oxide synthase (eNOS) and p22phox subunit of NAD(P)H oxidase was determined in aorta. Glutathione peroxidase activity was lower in SHR than in WKY rats, and LC increased this activity in SHR up to values close to those observed in normotensive animals. Glutathione reductase and catalase activities, which were higher in SHR, tended to increase after LC treatment. No differences were found in the activity of superoxide dismutase among any animal group. The ratio between reduced and oxidized glutathione and the levels of lipid peroxidation were respectively decreased and increased in hypertensive rats, and both parameters were normalized after the treatment. Similarly, LC was able to reverse the reduced plasma nitrite and nitrate levels and TAS observed in SHR. We found no alterations in the expression of aortic eNOS among any group; however, p22phox mRNA levels showed an increase in SHR that was reversed by LC. In conclusion, chronic administration of LC leads to an increase in hepatic and cardiac antioxidant defense and a reduction in the systemic oxidative process in SHR. Therefore, LC might increase NO availability in SHR aorta by a reduction in superoxide anion production.

l-Carnitine ameliorates the oxidative stress response to angiotensin II by modulating NADPH oxidase through a reduction in protein kinase c activity and NF-κB translocation to the nucleus.

l-Carnitine (LC) exerts beneficial effects in arterial hypertension due, in part, to its antioxidant capacity. We investigated the signalling pathways involved in the effect of LC on angiotensin II (Ang II)-induced NADPH oxidase activation in NRK-52E cells. Ang II increased the generation of superoxide anion from NADPH oxidase, as well as the amount of hydrogen peroxide and nitrotyrosine. Co-incubation with LC managed to prevent these alterations and also reverted the changes in NADPH oxidase expression triggered by Ang II. Cell signalling studies evidenced that LC did not modify Ang II-induced phosphorylation of Akt, p38 MAPK or ERK1/2. On the other hand, a significant decrease in PKC activity, and inhibition of nuclear factor kappa B (NF-kB) translocation, were attributable to LC incubation. In conclusion, LC counteracts the pro-oxidative response to Ang II by modulating NADPH oxidase enzyme via reducing the activity of PKC and the translocation of NF-kB to the nucleus.

Antioxidant activity of propionyl-L-carnitine in liver and heart of spontaneously hypertensive rats.

Oxidative stress plays an important role in arterial hypertension and propionyl-L-carnitine (PLC) has been found to protect cells from toxic reactive oxygen species. In this work, we have evaluated the antioxidant capacity of chronic PLC treatment in spontaneously hypertensive rats (SHR) by measuring the activity of antioxidant enzymes and the lipid peroxidation in liver and cardiac tissues. The activity of glutathione peroxidase was decreased in liver and cardiac tissues of SHR when compared with their normotensive controls, Wistar- Kyoto (WKY) rats, this alteration being prevented by PLC treatment. Glutathione reductase activity was increased in hypertensive rats and no effect was observed after the treatment. No significant changes in superoxide dismutase activity were observed among all experimental groups. Liver of hypertensive rats showed higher catalase activity than that of normotensive rats, and PLC enhanced this activity in both rat strains. Thiobarbituric acid reactive substances, determined as a measure of lipid peroxidation, were increased in SHR compared with WKY rats, and PLC treatment decreased these values not only in hypertensive rats but also in normotensive ones. The content of carnitine in serum, liver and heart was higher in PLC-treated rats, but PLC did not prevent the hypertension development in young SHR. In addition, triglyceride levels, which were lower in SHR than WKY rats, were reduced by chronic PLC treatment in both rat strains. These results demonstrate: i) the hypotriglyceridemic effect of PLC and ii) the antioxidant capacity of PLC in SHR and its beneficial use protecting tissues from hypertension-accompanying oxidative damage.

Regional difference in inflammatory response to LPS-injection in the brain: role of microglia cell density.

To elucidate whether density of cells could contribute to the extent of microglial activation, we performed in vitro assays using three different densities of N13 microglia stimulated with LPS. Our results showed that induction of pro-inflammatory factors as TNF-α and iNOS was directly related to cell density, meanwhile the induction of the anti-inflammatory IL-10 was inversely related to cell density. Accordingly, in vivo assays showed that after LPS-injection, iNOS expression was more intense in substantia nigra, a brain area showing specific susceptibility to neurodegeneration after microglia activation, whereas IL-10 expression was more sustained in striatum, an area resistant to damage. These results support that microglia density is pivotal to control the balance between pro- and anti-inflammatory factors release.

Molecular and cellular characterization of the age-related neuroinflammatory processes occurring in normal rat hippocampus: potential relation with the loss of somatostatin GABAergic neurons.

Increased neuroinflammatory reaction is frequently observed during normal brain aging. However, a direct link between neuroinflammation and neurodegeneration during aging has not yet been clearly shown. Here, we have characterized the age-related hippocampal inflammatory processes and the potential relation with hippocampal neurodegeneration. The mRNA expression of the pro-inflammatory cytokines IL-1beta and tumor necrosis factor-alpha (TNF-alpha), and the iNOs enzyme was significantly increased in aged hippocampus. Accordingly, numerous activated microglial cells were observed in aged rats. These cells were differentially distributed along the hippocampus, being more frequently located in the hilus and the CA3 area. The mRNA expression of somatostatin, a neuropeptide expressed by some GABAergic interneurons, and the number of somatostatin-immunopositive cells decreased in aged rats. However, the number of hippocampal parvalbumin-containing GABAergic interneurons was preserved. Interestingly, in aged rats, the mRNA expression of somatostatin and IL-1beta was inversely correlated and, the decrease in the number of somatostatin-immunopositive cells was higher in the hilus of dentate gyrus than in the CA1 region. Finally, intraperitoneal chronic lipopolysaccharide (LPS) injection in young animals mimicked the age-related hippocampal inflammation as well as the decrease of somatostatin mRNA expression. Present results strongly support the neuroinflammation as a potential factor involved in the age-related degeneration of somatostatin GABAergic cells.