Alessandra Stacchiotti

Long-Term Treatment with the Apolipoprotein A1 Mimetic Peptide Increases Antioxidants and Vascular Repair in Type I Diabetic Rats

Apolipoprotein A1 mimetic peptide (D-4F), synthesized from D-amino acid, enhances the ability of high-density lipoprotein to protect low-density lipoprotein (LDL) against oxidation in atherosclerotic disease. Using a rat model of type I diabetes, we investigated whether chronic use of D-4F would lead to up-regulation of heme oxygenase (HO)-1, endothelial cell marker (CD31+), and thrombomodulin (TM) expression and increase the number of endothelial progenitor cells (EPCs). Sprague-Dawley rats were rendered diabetic with streptozotocin (STZ) and either D-4F or vehicle was administered, by i.p. injection, daily for 6 weeks (100 μg/100 g b.wt.). HO activity was measured in liver, kidney, heart, and aorta. After 6 weeks of D-4F treatment, HO activity significantly increased in the heart and aorta by 29 and 31% (p < 0.05 and p < 0.49), respectively. Long-term D-4F treatment also caused a significant increase in TM and CD31+ expression. D-4F administration increased antioxidant capacity, as reflected by the decrease in oxidized protein and oxidized LDL, and enhanced EPC function and/or repair, as evidenced by the increase in EPC endothelial nitric-oxide synthase (eNOS) and prevention of vascular TM and CD31+ loss. In conclusion, HO-1 and eNOS are relevant targets for D-4F and may contribute to the D-4F-mediated increase in TM and CD31+, the antioxidant and anti-inflammatory properties, and confers robust vascular protection in this animal model of type 1 diabetes.

Stress proteins and oxidative damage in a renal derived cell line exposed to inorganic mercury and lead

A close link between stress protein up-regulation and oxidative damage may provide a novel therapeutic tool to counteract nephrotoxicity induced by toxic metals in the human population, mainly in children, of industrialized countries. Here we analysed the time course of the expression of several heat shock proteins, glucose-regulated proteins and metallothioneins in a rat proximal tubular cell line (NRK-52E) exposed to subcytotoxic doses of inorganic mercury and lead. Concomitantly, we used morphological and biochemical methods to evaluate metal-induced cytotoxicity and oxidative damage. In particular, as biochemical indicators of oxidative stress we detected reactive oxygen species (ROS) and nitrogen species (RNS), total glutathione (GSH) and glutathione-S-transferase (GST) activity. Our results clearly demonstrated that mercury increases ROS and RNS levels and the expressions of Hsp25 and inducible Hsp72. These findings are corroborated by evident mitochondrial damage, apoptosis or necrosis. By contrast, lead is unable to up-regulate Hsp72 but enhances Grp78 and activates nuclear Hsp25 translocation. Furthermore, lead causes endoplasmic reticulum (ER) stress, vacuolation and nucleolar segregation. Lastly, both metals stimulate the over-expression of MTs, but with a different time course. In conclusion, in NRK-52E cell line the stress response is an early and metal-induced event that correlates well with the direct oxidative damage induced by mercury. Indeed, different chaperones are involved in the specific nephrotoxic mechanism of these environmental pollutants and work together for cell survival.

Dose-dependent Mercuric Chloride Tubular Injury in Rat Kidney

Mercuric chloride (HgCl 2 ) produces an acute renal failure in experimental animal models. Since the mechanism of tubular injury has not completely been clarified, this morpho-quantitative study was undertaken to better understand the effects of 2 sublethal doses (T1=1 mg/kg and T3.5=3.5 mg/kg) of HgCl 2 in rat proximal tubules. Morphometrical analysis was performed to quantify both cytoplasmic and nuclear changes found in treated in respect to saline-injected proximal tubules. In the controls, single-cell damage was occasional and nucleolar changes were absent. HgCl 2 induced progressively severe proximal tubule atrophy. In the T1 group, necrosis was limited to pars recta cells and nucleolar segregation was often partial. In the T3.5 group, atrophy was extensive in both convoluted and straight tracts, the nucleolus was completely segregated and coiled body-like inclusions were detached from it. Ultrastructural analysis confirmed dose-dependent changes within straight proximal tubules, i.e., necrosis, apoptosis, nucleolar segregation, swollen mitochondria, vacuolization, and disrupted brush border. In conclusion, in the rat kidney HgCl 2 induced dose-dependent alterations not only in the cytoplasm but also in the nucleus of proximal tubule cells. These findings will be useful for better understanding of the pathogenesis of mercury nephrotoxicity and its genotoxic effect.

Melatonin reduces obesity and restores adipokine patterns and metabolism in obese (ob/ob) mice

The increasing incidence of obesity, leading to metabolic complications, is now recognized as a major public health problem. The adipocytes are not merely energy-storing cells, but they play crucial roles in the development of the so-called metabolic syndrome due to the adipocyte-derived bioactive factors such as adipokines, cytokines, and growth factors. The dysregulated production and secretion of adipokines seen in obesity is linked to the pathogenesis of the metabolic disease processes. In this study, we hypothesized that dietary melatonin administration would support an anti-inflammatory response and play an important role in energy metabolism in subcutaneous and visceral adipose tissues of obese mice and so may counteract some of the disruptive effects of obesity. Lean and obese mice (ob/ob) received melatonin or vehicle in drinking water for 8 weeks. Thereafter, they were evaluated for morphologic alteration, inflammatory cell infiltration, and the adipokine patterns in visceral and subcutaneous white fat depots. In obese mice treated with vehicle, we observed a significant increase in fat depots, inflammation, and a dysregulation of the adipokine network. In particular, we measured a significant reduction of adiponectin and an increase of tumor necrosis factor α, resistin, and visfatin in adipose tissue deposits. These changes were partially reversed when melatonin was supplemented to obese mice. Melatonin supplementation by regulating inflammatory infiltration ameliorates obesity-induced adipokine alteration, whereas melatonin administration in lean mice was unaffected. Thus, it is likely that melatonin would be provided in supplement form to control some of the disruptive effects on the basis of obesity pathogenic process.

Mitochondrial and metabolic dysfunction in renal convoluted tubules of obese mice: protective role of melatonin.

Obesity is a common and complex health problem, which impacts crucial organs; it is also considered an independent risk factor for chronic kidney disease. Few studies have analyzed the consequence of obesity in the renal proximal convoluted tubules, which are the major tubules involved in reabsorptive processes. For optimal performance of the kidney, energy is primarily provided by mitochondria. Melatonin, an indoleamine and antioxidant, has been identified in mitochondria, and there is considerable evidence regarding its essential role in the prevention of oxidative mitochondrial damage. In this study we evaluated the mechanism(s) of mitochondrial alterations in an animal model of obesity (ob/ob mice) and describe the beneficial effects of melatonin treatment on mitochondrial morphology and dynamics as influenced by mitofusin-2 and the intrinsic apoptotic cascade. Melatonin dissolved in 1% ethanol was added to the drinking water from postnatal week 5–13; the calculated dose of melatonin intake was 100 mg/kg body weight/day. Compared to control mice, obesity-related morphological alterations were apparent in the proximal tubules which contained round mitochondria with irregular, short cristae and cells with elevated apoptotic index. Melatonin supplementation in obese mice changed mitochondria shape and cristae organization of proximal tubules, enhanced mitofusin-2 expression, which in turn modulated the progression of the mitochondria-driven intrinsic apoptotic pathway. These changes possibly aid in reducing renal failure. The melatonin-mediated changes indicate its potential protective use against renal morphological damage and dysfunction associated with obesity and metabolic disease.

Schisandrin B stimulates a cytoprotective response in rat liver exposed to mercuric chloride

Mercury represents an ubiquitous environmental toxic metal. Heat shock proteins (HSP) and metallothioneins (MTs) help to protect cells against metal toxicity. Schisandrin B (Sch B), a lignoid from Schisandra chinensis, has been successfully used to treat hepatitis, but its effect against mercury hepatotoxicity remains unknown. We analyzed whether Sch B could protect rat liver against mercuric chloride (HgCl2) intake by analyzing stress proteins and histopathological changes. Wistar rats were administered Sch B (10mg/kg/day by gavage) or vehicle (olive oil) for 10 days. A subset of each group also received low-dose HgCl2 (0.1mg/kg/day) for 3 days on days 8-10. Another group received Sch B for 10 days with a single high dose of HgCl2 (1mg/kg intraperitoneally) on day 10. In rats treated with Sch B and HgCl2, HSP72, HSP25 and MTs were overexpressed in liver zones 1 and 3 irrespective of HgCl2 dosing schedules. Furthermore Sch B alone induced perinuclear rough endoplasmic reticulum alignment and if associated to HgCl2, increased mitochondrial density and dense bodies, all signs of intense detoxification machinery. Taking together these data suggest that dietary Sch B counteracts HgCl2 hepatotoxicity in the rat by stimulating chaperones responsible for anabolic activity.

Tubular Stress Proteins and Nitric Oxide Synthase Expression in Rat Kidney Exposed to Mercuric Chloride and Melatonin

Stress proteins such as HSP70 members (HSP72 and GRP75) and metallothionein (MT) protect the kidney against oxidative damage and harmful metals, whereas inducible nitric oxide synthase (iNOS) regulates tubular functions. A single dose of mercuric chloride (HgCl2) can cause acute renal failure in rats, its main target being the proximal tubule. Oxidative damage has been proposed as one of its pathogenic mechanisms. In this study we tested whether melatonin (MEL), a powerful antioxidant compound, is effective against HgCl2 nephrotoxicity. Rats were treated with saline, HgCl2 (3.5 mg/kg), MEL (5 mg/kg), and MEL + HgCl2 and examined after 24 hr for HSP72, GRP75, MT, and iNOS by immunohistochemistry and immunoblotting. Tubular effects of the treatment were then characterized by ultrastructure. In the HgCl2 group, all markers were overexpressed in convoluted proximal tubules and sometimes in distal tubules. In the MEL + HgCl2 group, GRP75 and iNOS decreased in convoluted and straight proximal tubules, whereas HSP72 and MT persisted more than the saline and MEL-only groups. Tubular damage and mitochondrial morphometry were improved by MEL pretreatment. In conclusion, the beneficial effect of MEL against HgCl2 nephrotoxicity was outlined morphologically and by the reduction of the tubular expression of stress proteins and iNOS. These markers could represent sensitive recovery index against mercury damage. (J Histochem Cytochem 54:1149-1157, 2006)

Hepatic Macrosteatosis Is Partially Converted to Microsteatosis by Melatonin Supplementation in ob/ob Mice Non-Alcoholic Fatty Liver Disease.

Background Obesity is a common risk factor for non-alcoholic fatty liver disease (NAFLD). Currently, there are no specific treatments against NAFLD. Thus, examining any molecule with potential benefits against this condition emerged melatonin as a molecule that influences metabolic dysfunctions. The aim of this study was to determine whether melatonin would function against NAFDL, studying morphological, ultrastuctural and metabolic markers that characterize the liver of ob/ob mice. Methods Lean and ob/ob mice were supplemented with melatonin in the drinking water for 8 weeks. Histology and stereology were performed to assess hepatic steatosis and glycogen deposition. Ultrastructural features of mitochondria, endoplasmic reticulum (ER) and their juxtapositions were evaluated in livers of all experimental groups. Furthermore, hepatic distribution and expression of markers of ER and mitochondria (calnexin, ATP sintase β, GRP78 and CHOP) and metabolic dysfunction (RPB4, β-catenin) and cellular longevity (SIRT1) were analyzed. Results Melatonin significantly reduced glycemia, identified also by a decrease of hepatic RBP4 expression, reversed macrosteatosis in microsteatosis at the hepatic pericentral zone, enlarged ER-mitochondrial distance and ameliorated the morphology and organization of these organelles in ob/ob mouse liver. Furthermore, in ob/ob mice, calnexin and ATP synthase β were partially restored, GRP78 and CHOP decreased in periportal and midzonal hepatocytes and β-catenin expression was, in part, restored in peripheral membranes of hepatocytes. Melatonin supplementation to ob/ob mice improves hepatic morphological, ultrastructural and metabolic damage that occurs as a result of NAFLD. Conclusions Melatonin may be a potential adjuvant treatment to limit NAFLD and its progression into irreversible complications.

Endothelial and vascular smooth muscle cell dysfunction mediated by cyclophylin A and the atheroprotective effects of melatonin

AIMS This study evaluated the role of cyclophilin A (CyPA) in early phase of atherosclerosis and also examined the atheroprotective effects of melatonin due to its antioxidant properties. MAIN METHODS APOE null mice at 6 and 15weeks of age were treated with melatonin at a dose of 0.1mg/kg/day or 10mg/kg/day. We evaluated both histopathological alterations in endothelial and vascular smooth muscle cells by CyPA and rolling mononuclear cell expression during the early phase of atherosclerosis development. KEY FINDINGS Our study showed that CyPA expression increases and may modulate inflammatory cell adhesion and interleukin-6 expression inducing vascular smooth muscle cell migration and inflammatory cell extravasation in a time-dependent manner. Moreover, we observed an indirect atheroprotective effect of melatonin on vascular injury; it inhibited CyPA mediated inflammatory cell extravasation and oxidative stress. SIGNIFICANCE The melatonin treatment may represent a new atheroprotective approach that contributes to reducing the early phase of atherosclerosis involving the rolling of monocytes, their passage to subendothelial space and inhibition of CyPA expression.

Different role of Schisandrin B on mercury-induced renal damage in vivo and in vitro

Mercuric chloride (HgCl₂) causes acute oxidant renal failure that affects mainly proximal tubules. Schisandrin B (Sch B), an active lignan from the fruit of Schisandra chinensis, has been successfully used to treat gentamicin nephrotoxicity, but its role against mercury damage is still largely unknown. Here we analysed in vivo and in vitro the efficacy of Sch B supplementation against HgCl₂ nephrotoxicity, focusing on histopathology, stress proteins, oxidative (cytochrome c oxidase) and nitrosactive markers (eNOS, nNOS). Wistar rats were treated with Sch B (10 mg/kg/day p.o.) or vehicle (olive oil) for 9 days, then coadministered with a single HgCl₂ nephrotoxic dose (3.5 mg/kg i.p.) and killed after 24 h. The tubular and mitochondrial damage induced by mercury was limited by Sch B coadministration in vivo. Remarkably, after Sch B and mercury challenge, HSP25, HSP72, GRP75 were reduced in the renal cortex, cytochrome c oxidase increased and eNOS and nNOS were restored in glomeruli. In contrast, NRK-52E proximal tubular cells treated with Sch B 6.25 μM plus HgCl₂ 20 μM did not show any amelioration on viability and oxidative stress in respect to HgCl₂ 20 μM alone. Moreover, after Sch B plus mercury in vitro treatment, HSP72 staining persisted while HSP25 further increased. Thus, in our experimental conditions, Sch B cotreatment afforded better protection against mercury poisoning in vivo than in vitro. This discrepancy might be partly attributable to Sch B influence on glomerular perfusion as corroborated by the recovery of vasoactive markers like macular and endothelial nitric oxide isoforms.