Freddy Romero

Melatonin prevents oxidative stress resulting from iron and erythropoietin administration

Intravenous iron (Fe) and recombinant human erythropoietin (rHuEPO) are routine treatments in the management of anemia in patients with chronic renal failure. We investigated the oxidative stress acutely induced by these therapies and whether pretreatment with oral melatonin (MEL) would have a beneficial effect. Nine patients (four women) were studied within 1 month of entering a chronic hemodialysis program in the interdialytic period. Plasma malondialdehyde (MDA), red blood cell glutathione (GSH), and catalase (CAT) activity were measured in blood samples obtained before (baseline) and 1, 3, and 24 hours after the administration of Fe (100 mg of Fe saccharate intravenously over 1 hour) or rHuEPO (4,000 U intravenously). One hour before these treatments, patients were administered a single oral dose of MEL (0.3 mg/kg) or placebo. Each patient was studied on four occasions, corresponding to studies performed using either placebo or MEL in association with intravenous Fe and rHuEPO administration. Baseline data showed increased oxidative stress in patients with end-stage renal failure. Increments in oxidative stress induced by Fe were more pronounced at the end of the administration: MDA, baseline, 0.74 +/- 0.09 nmol/mL; 1 hour, 1.50 +/- 0.28 nmol/mL (P: < 0.001); GSH, baseline, 2.51 +/- 0.34 nmol/mg of hemoglobin (Hb); 1 hour, 1.66 +/- 0.01 nmol/mg Hb (P: < 0.001); and CAT activity, baseline, 27.0 +/- 5.7 kappa/mg Hb; 1 hour, 23.3 +/- 4.2 kappa/mg Hb (P: < 0.001). rHuEPO-induced increments in oxidative stress were more pronounced (P: < 0.001) at 3 hours (MDA, 1.24 +/- 0.34 nmol/mL; GSH, 1.52 +/- 0.23 nmol/mg Hb; CAT activity, 18.0 +/- 3.1 kappa/mg Hb). MEL administration prevented the changes induced by Fe and rHuEPO and had no adverse side effects. These studies show that intravenous Fe and rHuEPO in doses commonly used to treat anemia in chronic hemodialysis patients acutely generate significant oxidative stress. Oral MEL prevents such oxidative stress and may be of clinical use.

Effect of Uric Acid on Gentamicin‐Induced Nephrotoxicity in Rats – Role of Matrix Metalloproteinases 2 and 9

In this work, we aimed to study the effect of uric acid on gentamicin-induced nephrotoxicity. Male Sprague-Dawley rats were assigned to one of six groups (six rats each) which received intraperitoneal injections for 9 days: (S) saline; (UA) Uric acid alone; (G) Gentamicin alone; (G + UA) Gentamicin + uric acid; (G rec) Gentamicin recovery and (G + UA rec) Gentamicin + uric acid recovery. In (G rec) and (G + UA rec), rats recovered for 7 days after the last injection. Urine and blood samples were taken on day 0 and at the end of every stage. Kidneys were harvested for histological scoring, determination of renal malondialdehyde (MDA), zymography and western blots for matrix metalloprotease (MMP)-2 and MMP-9. Uric acid alone did not provoke changes in biochemical and histological parameters when compared to controls. Gentamicin alone increased significantly plasma creatinine and blood urea nitrogen and caused a moderate histological damage. When combined with uric acid, these conditions worsened. MMP-9 activity and expression was decreased in rats from group G + UA as compared with rats from group G, while activity of MMP-2 was similarly increased in both groups when compared to controls. The increase in renal MDA induced by gentamicin was not altered when it was combined with uric acid. During the recovery stage, all biochemical parameters returned to normal levels, though a trend for delay of tubular damage recovery was observed in group G + UA rec when compared with group G rec. The results indicate that uric acid worsens gentamicin-induced nephrotoxicity. The mechanism is likely to implicate down-regulation of MMP-9.

Obesity-induced adipokine imbalance impairs mouse pulmonary vascular endothelial function and primes the lung for injury

Obesity is a risk factor for the development of acute respiratory distress syndrome (ARDS) but mechanisms mediating this association are unknown. While obesity is known to impair systemic blood vessel function, and predisposes to systemic vascular diseases, its effects on the pulmonary circulation are largely unknown. We hypothesized that the chronic low grade inflammation of obesity impairs pulmonary vascular homeostasis and primes the lung for acute injury. The lung endothelium from obese mice expressed higher levels of leukocyte adhesion markers and lower levels of cell-cell junctional proteins when compared to lean mice. We tested whether systemic factors are responsible for these alterations in the pulmonary endothelium; treatment of primary lung endothelial cells with obese serum enhanced the expression of adhesion proteins and reduced the expression of endothelial junctional proteins when compared to lean serum. Alterations in pulmonary endothelial cells observed in obese mice were associated with enhanced susceptibility to LPS-induced lung injury. Restoring serum adiponectin levels reversed the effects of obesity on the lung endothelium and attenuated susceptibility to acute injury. Our work indicates that obesity impairs pulmonary vascular homeostasis and enhances susceptibility to acute injury and provides mechanistic insight into the increased prevalence of ARDS in obese humans.

Role of Angiotensin II type 1 receptor on renal NAD(P)H oxidase, oxidative stress and inflammation in nitric oxide inhibition induced-hypertension

AIMS Activation of the renin-angiotensin system (RAS), renal oxidative stress and inflammation are constantly present in experimental hypertension. Nitric oxide (NO) inhibition with N(w)-nitro-L-arginine methyl ester (L-NAME) has previously been reported to produce hypertension, increased expression of Angiotensin II (Ang II) and renal dysfunction. The use of Losartan, an Ang II type 1 receptor (AT1R) antagonist has proven to be effective reducing hypertension and renal damage; however, the mechanism by which AT1R blockade reduced kidney injury and normalizes blood pressure in this experimental model is still complete unknown. The current study was designed to test the hypothesis that AT1R activation promotes renal NAD(P)H oxidase up-regulation, oxidative stress and cytokine production during L-NAME induced-hypertension. MAIN METHODS Male Sprague-Dawley rats were distributed in three groups: L-NAME, receiving 70 mg/100ml of L-NAME, L-NAME+Los, receiving 70 mg/100ml of L-NAME and 40 mg/kg/day of Losartan; and Controls, receiving water instead of L-NAME or L-NAME and Losartan. KEY FINDINGS After two weeks, L-NAME induced high blood pressure, renal overexpression of AT1R, NAD(P)H oxidase sub-units gp91, p22 and p47, increased levels of oxidative stress, interleukin-6 (IL-6) and interleukin-17 (IL-17). Also, we found increased renal accumulation of lymphocytes and macrophages. Losartan treatment abolished the renal expression of gp91, p22, p47, oxidative stress and reduced NF-κB activation and IL-6 expression. SIGNIFICANCE These findings indicate that NO induced-hypertension is associated with up-regulation of NADPH oxidase, oxidative stress production and overexpression of key inflammatory mediators. These events are associated with up-regulation of AT1R, as evidenced by their reversal with AT1R blocker treatment.

Isolation and cloning of the K+-independent, ouabain-insensitive Na+-ATPase

Primary Na+ transport has been essentially attributed to Na+/K+ pump. However, there are functional and biochemical evidences that suggest the existence of a K+-independent, ouabain-insensitive Na+ pump, associated to a Na+-ATPase with similar characteristics, located at basolateral plasma membrane of epithelial cells. Herein, membrane protein complex associated with this Na+-ATPase was identified. Basolateral membranes from guinea-pig enterocytes were solubilized with polyoxyethylene-9-lauryl ether and Na+-ATPase was purified by concanavalin A affinity and ion exchange chromatographies. Purified enzyme preserves its native biochemical characteristics: Mg2+ dependence, specific Na+ stimulation, K+ independence, ouabain insensitivity and inhibition by furosemide (IC50: 0.5 mM) and vanadate (IC50: 9.1 μM). IgY antibodies against purified Na+-ATPase did not recognize Na+/K+-ATPase and vice versa. Analysis of purified Na+-ATPase by SDS-PAGE and 2D-electrophoresis showed that is constituted by two subunits: 90 (α) and 50 (β) kDa. Tandem mass spectrometry of α-subunit identified three peptides, also present in most Na+/K+-ATPase isoforms, which were used to design primers for cloning both ATPases by PCR from guinea-pig intestinal epithelial cells. A cDNA fragment of 1148 bp (atna) was cloned, in addition to Na+/K+-ATPase α1-isoform cDNA (1283 bp). In MDCK cells, which constitutively express Na+-ATPase, silencing of atna mRNA specifically suppressed Na+-ATPase α-subunit and ouabain-insensitive Na+-ATPase activity, demonstrating that atna transcript is linked to this enzyme. Guinea-pig atna mRNA sequence (2787 bp) was completed using RLM-RACE. It encodes a protein of 811 amino acids (88.9 kDa) with the nine structural motifs of P-type ATPases. It has 64% identity and 72% homology with guinea-pig Na+/K+-ATPase α1-isoform. These structural and biochemical evidences identify the K+-independent, ouabain-insensitive Na+-ATPase as a unique P-type ATPase.

Cavin1; a Regulator of Lung Function and Macrophage Phenotype

Caveolae are cell membrane invaginations that are highly abundant in adipose tissue, endothelial cells and the lung. The formation of caveolae is dependent on the expression of various structural proteins that serve as scaffolding for these membrane invaginations. Cavin1 is a newly identified structural protein whose deficiency in mice leads to loss of caveolae formation and to development of a lipodystrophic phenotype. In this study, we sought to investigate the functional role of Cavin1 in the lung. Cavin1 deficient mice possessed dramatically altered distal lung morphology and exhibited significant physiological alterations, notably, increased lung elastance. The changes in distal lung architecture were associated with hypercellularity and the accumulation of lung macrophages. The increases in lung macrophages occurred without changes to circulating numbers of mononuclear cells and without evidence for increased proliferation. However, the increases in lung macrophages were associated with higher levels of macrophage chemotactic factors CXCL2 and CCL2 in BAL fluid from Cavin1−/− mice suggesting a possible mechanism by which these cells accumulate. In addition, lung macrophages from Cavin1−/− mice were larger and displayed measurable differences in gene expression when compared to macrophages from wild-type mice. Interestingly, macrophages were also increased in adipose tissue but not in liver, kidney or skeletal muscle from Cavin1−/− mice, and similar tissue specificity for macrophage accumulation was observed in lungs and adipose tissue from Caveolin1−/− mice. In conclusion, this study demonstrates an important role for Cavin1 in lung homeostasis and suggests that caveolae structural proteins are necessary for regulating macrophage number and phenotype in the lung.

IL-15Rα is a determinant of muscle fuel utilization, and its loss protects against obesity.

IL-15Rα is the widely expressed primary binding partner for IL-15. Because of the wide distribution in nonlymphoid tissues like skeletal muscle, adipose, or liver, IL-15/IL-15Rα take part in physiological and metabolic processes not directly related to immunity. In fast muscle, lack of IL-15Rα promotes an oxidative switch, with increased mitochondrial biogenesis and fatigue resistance. These effects are predicted to reproduce some of the benefits of exercise and, therefore, improve energy homeostasis. However, the direct effects of IL-15Rα on metabolism and obesity are currently unknown. We report that mice lacking IL-15Rα (IL-15Rα(-/-)) are resistant to diet-induced obesity (DIO). High-fat diet-fed IL-15Rα(-/-) mice have less body and liver fat accumulation than controls. The leaner phenotype is associated with increased energy expenditure and enhanced fatty acid oxidation by muscle mitochondria. Despite being protected against DIO, IL-15Rα(-/-) are hyperglycemic and insulin-resistant. These findings identify novel roles for IL-15Rα in metabolism and obesity.

Chronic Alcohol Ingestion in Rats Alters Lung Metabolism, Promotes Lipid Accumulation, and Impairs Alveolar Macrophage Functions

Chronic alcoholism impairs pulmonary immune homeostasis and predisposes to inflammatory lung diseases, including infectious pneumonia and acute respiratory distress syndrome. Although alcoholism has been shown to alter hepatic metabolism, leading to lipid accumulation, hepatitis, and, eventually, cirrhosis, the effects of alcohol on pulmonary metabolism remain largely unknown. Because both the lung and the liver actively engage in lipid synthesis, we hypothesized that chronic alcoholism would impair pulmonary metabolic homeostasis in ways similar to its effects in the liver. We reasoned that perturbations in lipid metabolism might contribute to the impaired pulmonary immunity observed in people who chronically consume alcohol. We studied the metabolic consequences of chronic alcohol consumption in rat lungs in vivo and in alveolar epithelial type II cells and alveolar macrophages (AMs) in vitro. We found that chronic alcohol ingestion significantly alters lung metabolic homeostasis, inhibiting AMP-activated protein kinase, increasing lipid synthesis, and suppressing the expression of genes essential to metabolizing fatty acids (FAs). Furthermore, we show that these metabolic alterations promoted a lung phenotype that is reminiscent of alcoholic fatty liver and is characterized by marked accumulation of triglycerides and free FAs within distal airspaces, AMs, and, to a lesser extent, alveolar epithelial type II cells. We provide evidence that the metabolic alterations in alcohol-exposed rats are mechanistically linked to immune impairments in the alcoholic lung: the elevations in FAs alter AM phenotypes and suppress both phagocytic functions and agonist-induced inflammatory responses. In summary, our work demonstrates that chronic alcohol ingestion impairs lung metabolic homeostasis and promotes pulmonary immune dysfunction. These findings suggest that therapies aimed at reversing alcohol-related metabolic alterations might be effective for preventing and/or treating alcohol-related pulmonary disorders.

Effect of uric acid on nephrotoxicity induced by mercuric chloride in rats

Oxidative stress is an important mechanism in mercury poisoning. We studied the effect of uric acid, a natural and potent reactive oxygen species and peroxynitrite scavenger, in HgCl 2-induced nephrotoxicity. Rats were injected with a unique dose of HgCl2 (2.5 mg/kg body weight, subcutaneously) and then vehicle (for 3 days, twice daily) or HgCl2 (unique dose) and intraperitoneal uric acid suspension (250 mg/kg body weight, twice daily, for 3 days), and then killed at 24, 48 and 72 hours after HgCl2 administration (n = 5 for each group). At the end of the experimental study, kidneys and blood samples were taken. Tissues were prepared and examined under light microscopy. Uric acid significantly prevented the increase in plasma levels of creatinine and blood urea nitrogen (BUN); it helped maintain systemic nitrate/nitrite concentration and total antioxidant capacity. Uric acid attenuated the increase of renal lipid peroxidation and it markedly diminished nitrotyrosine signal and histopathological changes as early as 24 hours after HgCl2 administration. Uric acid did not prevent a decrease in β-actin signal caused by mercuric chloride, but it promoted a faster recovery when compared to the HgCl2 alone group. Our results indicate that UA could play a beneficial role against HgCl2 toxicity by preventing systemic and renal oxidative stress and tissue damage.

Effect of uric acid on hypertension progression in spontaneously hypertensive rats.

AIMS We investigated the effects of soluble uric acid (UA) on the development of hypertension in spontaneously hypertensive rats (SHRs), using Wistar-Kyoto rats (WKY) as normotensive controls. MAIN METHODS UA was prepared freshly as a suspension in saline solution and administered twice daily, intraperitoneally, at a dose of 250 mg/kg body weight for five weeks to SHR-UA (n=7) and WKY-UA rats (n=5). Controls for both strains were injected with saline solution (WKY-C and SHR-C, n=5 each). Blood pressure, determined by tail-cuff plethysmography, levels of urine and blood biochemical parameters were monitored weekly. Oxidative stress, renal cytokines mRNA levels and immune cells infiltration were determined at the end of the study. KEY FINDINGS UA did not alter blood pressure in the WKY rats, but markedly prevented the development of hypertension in SHRs. Urine volume was significantly increased in the SHR-UA group. UA protected against renal oxidative stress as indicated by a decrease in MDA content in the SHR-UA group when compared to the SHR-C group; MDA content was unchanged in the WKY animals. Plasma antioxidant capacity decreased significantly in the SHR-UA animals when compared to the other three groups. There was a significant decrease in renal infiltrating lymphocytes in the SHR-UA treated animals. Changes in the expression of TNF-alpha, IL-2 and IL-6 were detected in the SHR-UA group. SIGNIFICANCE We conclude that UA protects against progression of hypertension in SHR rats.