Mechanisms underlying the cardiac antifibrotic effects of losartan metabolites.

Excessive myocardial collagen deposition and cross-linking (CCL), a process regulated by lysyl oxidase (LOX), determines left ventricular (LV) stiffness and dysfunction. The angiotensin II antagonist losartan, metabolized to the EXP3179 and EXP3174 metabolites, reduces myocardial fibrosis and LV stiffness in hypertensive patients. Our aim was to investigate the differential influence of losartan metabolites on myocardial LOX and CCL in an experimental model of hypertension with myocardial fibrosis, and whether EXP3179 and EXP3174 modify LOX expression and activity in fibroblasts. In rats treated with NG-nitro-L-arginine methyl ester (L-NAME), administration of EXP3179 fully prevented LOX, CCL and connective tissue growth factor (CTGF) increase, as well as fibrosis, without normalization of blood pressure (BP). In contrast, administration of EXP3174 normalized BP and attenuated fibrosis but did not modify LOX, CCL and CTGF. In TGF-ß1-stimulated fibroblasts, EXP3179 inhibited CTGF and LOX expression and activity with lower IC50 values than EXP3174. Our results indicate that, despite a lower antihypertensive effect, EXP3179 shows higher anti-fibrotic efficacy than EXP3174, likely through its ability to prevent the excess of LOX and CCL. It is suggested that the anti-fibrotic effect of EXP3179 may be partially mediated by the blockade of CTGF-induced LOX in fibroblasts.

A synthetic peptide from transforming growth factor-ß1 type III receptor inhibits NADPH oxidase and prevents oxidative stress in the kidney of spontaneously hypertensive rats.

AIMS: The NADPH oxidases constitute a major source of superoxide anion (·O2(-)) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by transforming growth factor-ß1 (TGF-ß1). We investigated whether a chronic treatment with P144, a peptide synthesized from type III TGF-ß1 receptor, inhibited NADPH oxidases in the renal cortex of spontaneously hypertensive rats (SHR). RESULTS: Here, we show that chronic administration of P144 significantly reduced the NADPH oxidase expression and activity as well as the oxidative stress observed in control vehicle-treated SHR (V-SHR). In addition, P144 was also able to reduce the significant increase in the renal fibrosis and in mRNA expression of different components of collagen metabolism, as well as in the levels of connective tissue growth factor observed in V-SHR. Finally, TGF-ß1-stimulated NRK52E exhibited a significant increase in NADPH oxidase expression and activity as well as a TGF-ß1-dependent intracellular pathway that were inhibited in the presence of P144. INNOVATION: Our experimental evidence suggests that reversing oxidative stress may be therapeutically useful in preventing fibrosis-associated renal damage. We show here that (i) the TGF-ß1-NADPH oxidases axis is crucial in the development of fibrosis in an experimental hypertensive renal disease animal model, and (ii) the use of P144 reverses TGF-ß1-dependent NADPH oxidase activity; thus, P144 may be considered a novel therapeutic tool in kidney disease associated with hypertension. CONCLUSION: We demonstrate that P144 inhibits NADPH oxidases and prevents oxidative stress in kidneys from hypertensive rats. Our data also suggest that these effects are associated with the renal antifibrotic effect of P144.

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.

Blockade of TGF-ß 1 signalling inhibits cardiac NADPH oxidase overactivity in hypertensive rats.

NADPH oxidases constitute a major source of superoxide anion (·O(2)(-)) in hypertension. Several studies suggest an important role of NADPH oxidases in different effects mediated by TGF-ß 1. In this study we show that chronic administration of P144, a peptide synthesized from type III TGF-ß 1 receptor, significantly reduced the cardiac NADPH oxidase expression and activity as well as in the nitrotyrosine levels observed in control spontaneously hypertensive rats (V-SHR) to levels similar to control normotensive Wistar Kyoto rats. In addition, P144 was also able to reduce the significant increases in the expression of collagen type I protein and mRNA observed in hearts from V-SHR. In addition, positive correlations between collagen expression, NADPH oxidase activity, and nitrotyrosine levels were found in all animals. Finally, TGF-ß 1-stimulated Rat-2 exhibited significant increases in NADPH oxidase activity that was inhibited in the presence of P144. It could be concluded that the blockade of TGF-ß 1 with P144 inhibited cardiac NADPH oxidase in SHR, thus adding new data to elucidate the involvement of this enzyme in the profibrotic actions of TGF-ß 1.

The A640G CYBA polymorphism associates with subclinical atherosclerosis in diabetes.

Oxidative stress is implicated in diabetes. The NADPH oxidases are the main source of superoxide in phagocytic and vascular cells, and p22phox is a key subunit. Genetic variants of CYBA, the human p22phox gene, associate with cardiovascular disease. We investigated the association of the A640G polymorphism with diabetes and its impact on phagocytic NADPH oxidase-dependent superoxide production and subclinical atherosclerosis. We studied 1212 subjects in which clinical parameters including carotid intima-media thickness (cIMT) were assessed. The A640G polymorphism was genotyped by TaqMan probes. In 496 subjects, the NADPH oxidase-dependent superoxide production in peripheral blood mononuclear cells was assessed by chemiluminescence. The GG genotype prevalence was significantly higher in type 2 diabetic patients than in non-diabetic subjects. Peripheral blood mononuclear cells from diabetic GG patients presented higher NADPH oxidase-dependent superoxide production than those of diabetic AA/AG patients. Within the diabetic group, GG patients presented higher cIMT levels than AA/AG patients. The A640G CYBA polymorphism may be a marker of oxidative stress risk and may be indicative of subclinical atherosclerosis in type 2 diabetes.

Subchronic effects of cyanobacterial cells on the transcription of antioxidant enzyme genes in tilapia (Oreochromis niloticus).

The increasing occurrence of toxic cyanobacterial blooms in eutrophic water bodies is nowadays of worldwide concern due to their ability to produce toxins such as microcystins (MCs). These cyanobacterial toxins have been shown to affect aquatic organisms such as fish, resulting in oxidative stress. Among the antioxidant enzymes, glutathione peroxidase (GPx) and soluble glutathione-S-transferases (sGST) play an important role in the detoxification of MCs. In the present work tilapia (Oreochromis niloticus) were orally exposed to cyanobacterial cells containing MCs and non-containing MCs for 21 days. The activity and relative mRNA expression by real-time PCR of both enzymes and the GST protein abundance by Western blot analysis were evaluated in liver and kidney. Also the induction of lipid peroxidation (LPO) was assayed. MCs containing cyanobacterial cells induced an increase of LPO products in both organs, and MCs containing and MCs non-containing cyanobacterial cells altered the activity, gene expression and protein abundance of the enzymes, indicating the importance of GPx and sGST in MCs detoxification. Moreover, liver, the main organ involved in biodegradation and biotransformation, experienced an adaptative response to the toxic insult. These results show for the first time that the subchronic exposure to cyanobacterial cells causes changes in antioxidant and detoxification enzymes and that GPx and GST gene expression are good markers of these alterations in tilapia.

Nitric oxide involved in the IL-1ß-induced inhibition of fructose intestinal transport.

Interleukin-1ß (IL-1ß) is a pleiotropic cytokine produced by cells of the immune system and a large variety of other cell types including endothelial cells. It is released during inflammatory and infectious diseases, and possesses a wide spectrum of autocrine, paracrine and endocrine activities. The aim of this work was to examine the IL-1ß effect on D-fructose transport across rabbit jejunum and try to identify the mediators implicated in this process. A sepsis condition was induced for 90 min after intravenous (iv) administration of IL-1ß and body temperature was recorded. Studies on cellular intestinal integrity have not shown modifications of the epithelium and the basement membrane. D-fructose intestinal transport was studied in rabbit jejunum from control and treated animals and it was reduced in the latter ones. This cytokine decreased both the mucosal to serosal transepithelial flux and the transport across brush-border membrane vesicles of D-fructose. The inhibition was reversed by L-NAME (nitric oxide [NO] synthase inhibitor), but not by indomethacin (cyclooxygenase 1 and 2 inhibitor). Both inhibitors were administered iv 15 min before the IL-1ß. The protein levels of GLUT5 were not changed in all animal groups and those of mRNA were even increased. In summary, these findings indicate that IL-1ß, at the time assayed, induced a significant reduction in the relative intrinsic activity of GLUT5 and in this decrease are involved NO signalling pathways. In this way, blockage of D-fructose intestinal uptake by IL-1ß may be playing an essential role in the pathophysiology of septic shock.