Cristina Filippi

Resveratrol, a component of wine and grapes, in the prevention of kidney disease

Abstract: Ischemia is an inciting factor in 50% of incidences of acute renal failure, and it increases the risk of organ rejection after renal transplantation. We have previously demonstrated that resveratrol (RSV) reduces ischemia‐reperfusion (I/R) injury of rat kidney both by antioxidant and anti‐inflammatory mechanisms. However, a clear morphological demonstration of this activity has not been made. To answer this question we have performed a new set of experiments following the experimental protocol reported below to investigate the effects of I/R injury and RSV pretreatment on kidney morphology by computerized morphometric analysis. Both renal arteries were clamped for 40 minutes in 40 male Wistar rats (b.w. 220 ± 20 g); 20 rats were pretreated with RSV 1 μM e.v. 40 minutes before clamping. All animals were reperfused for 24 hours and then sacrificed. Histological examination showed tissue conservation in treated rats. I/R‐induced glomerular collapse (as revealed by mean glomerular volume and glomerular shape factor) was significantly reduced by RSV pretreatment. Capillary tuft/Bowmans capsule area ratio was enhanced in the I/R group suggesting tubular hypertension. RSV pre‐treatments significantly reduced this parameter to the control value. The number of platelet clots in the capillary tuft and tubular necrosis were also reduced by RSV versus I/R group. l‐NAME administration worsened both functional and structural damage. Finally, cGMP urinary levels were markedly reduced from 12.1 ± 8.4 nmol/day to 0.10 ± 0.10 nmol/day in the I/R group. RSV provided cGMP (5.01 ± 1.5 nmol/day, P < 0.05). As expected, l‐NAME administration significantly reduced cGMP in urine (0.71 ± 0.6 nmol/day). The present study confirms the protective effect of RSV pretreatment in I/R injury of rat kidney and suggests multiple mechanisms of action.

Interleukin-8 is a powerful prognostic predictor of all-cause and cardiovascular mortality in dialytic patients.

Background: Cohort studies have demonstrated an association between C-reactive protein (CRP) and interleukin-6 (IL-6) and all-cause and cardiovascular mortality in end-stage renal disease (ESRD) patients. Interleukin-8 (IL-8) appears to be not only the plasma expression of the acute-phase response but also a direct pathogenetic mediator of the atherosclerotic process. Methods: To evaluate the role of IL-8 in predicting outcome, 76 chronic dialytic patients were prospectively followed for 18 months. At baseline, blood samples were taken for analysis of high-sensitivity CRP, IL-6, IL-8 and other standard laboratory analyses. Results: Median IL-8 was 5.2 mg/l, therefore near half of the patients had IL-8 values within the range of ‘normal limits’. IL-6 and CRP were significantly correlated (r = 0.45, p < 0.001) and a positive correlation was also found between IL-6 and IL-8 (r = 0.39, p < 0.001). The correlation coefficient between IL-6 and CRP was 0.43 (p < 0.001) and 0.50 (p < 0.001) in patients without and with history and/or clinical signs of cardiovascular disease, respectively. After a follow-up of 1.5 years, 8 patients had died from cardiovascular causes and another 7 patients for other reasons; furthermore 9 major nonfatal cardiovascular events were recorded. Stepwise regression analysis showed IL-8 as the strongest independent predictor of all-cause and cardiovascular events (p = 0.0025) even after adjustment for age and dialytic age, followed by IL-6 and CRP (p < 0.01). Conclusion: Despite a small population and a relatively short follow-up period, this study firstly demonstrated that IL-8 is a powerful independent predictive factor for cardiovascular and overall mortality cause in ESRD patients.

Melatonin prevents cyclosporine-induced nephrotoxicity in isolated and perfused rat kidney

Cyclosporine A (CsA) is a potent and effective immunosuppressive agent, but its action is frequently accompanied by severe renal toxicity. The precise mechanism by which CsA causes renal injury is not known. Reactive oxygen species (ROS) have been shown to play a role, since CsA-induced renal lipid peroxidation is attenuated in vivo and in vitro by the concomitant administration of antioxidants such as vitamin E. We show here the effect of the antioxidant melatonin (MLT), a hormone produced by the pineal gland during the dark phase of the circadian cycle, in a model of CsA nephrotoxicity in the isolated and perfused rat kidney. Kidneys isolated from rats were divided into seven groups. At the end of perfusion, malondialdehyde and 4-hydroxyalkenals (MDA+4-HDA), metabolites of nitric oxide N O 2 m +N O 3 m were measured and histopathological examination was performed. CsA treatment induced a significant increase in MDA+4-HDA while not affecting the nitric oxide metabolite level. MLT remarkably prevented glomerular collapse and tubular damage as revealed by morphometric analysis. Our study suggests that lipid peroxidation is an early important event in the pathogenesis of CsA nephrotoxicity and that MLT is able to protect kidneys from CsA at a relatively low concentration.

Toxic effects of aluminium, chromium and cadmium in intact and regenerating freshwater planarians

Abstract The effects of exposure to heavy metals were analyzed in the intact and regenerating planarian Dugesia etrusca. Al3+ and Cr3+ were lethal at 1.5 mM concentration. Intact specimens, treated with A1 at lower concentrations, exhibited a variety of sublethal responses; Al was significantly more toxic than Cr. Alterations in the cephalic regeneration of decapitated planarians were observed with both metals. Cd2+ was tested at concentrations ranging from 0.18 μM to 18 μM. Until 1.8 μM, this metal did not produce any detectable sublethal responses in intact planarians, whereas higher concentrations were lethal. Sublethal Cd concentrations did not show any visible effect on cephalic regeneration.

Nitric Oxide–Dependent Renal Vasodilatation Is Not Altered in Rat with rHuEpo–Induced Hypertension

Background: Recombinant human erythropoietin (rHuEpo) is the treatment of choice in anemia associated with end–stage renal disease. Its major side effect is hypertension, which occurs in 8–30% of uremic patients. The exact mechanism of rHuEpo–induced hypertension has not been fully elucidated, and several possibilities have been proposed, such as a direct vascular effect of the drug with a shift in the balance of constrictor and relaxing endothelial factors (endothelins and nitric oxide (NO)). Recent papers suggested an enhanced rather than reduced activity of endogenous NO system in rats with normal renal function and rHuEpo–induced hypertension. Our study was designed to verify whether, in spite of enhanced activity of the renal NO system, rHuEpo may affect endothelium–dependent (acetylcholine–induced) and/or endothelium–independent (sodium nitroprusside–induced) vasorelaxation and to evaluate basal NO release by the infusion of NG–nitro–L–arginine methyl ester (L–NAME) in an isolated and perfused rat kidney model. Methods: To investigate this hypothesis, we have determined systemic and renal NO activity in Wistar rats treated with a hypertensive dose of rHuEpo (150 IU/kg b.w. every other day for 2 weeks) by measuring stable NO metabolites (NO2+NO3) in the urine and have also evaluated variations in renal vascular resistance after the injection of Ach, SNP and the infusion of L–NAME. Results: Hematocrit, hemoglobin concentration and arterial blood pressure were significantly increased in the treated group as compared with the controls. Urinary excretion of NO2+NO3 was significantly higher in treated than in the controls (438±66 vs. 294±36 nM/ml/min, p<0.01, respectively). There were no significant differences in the dose–response curves to Ach and SNP between the two groups. The renal vasoconstriction following the infusion of L–NAME was also similar in the two groups. Conclusions: The analysis of our results seems to indicate that the endogenous NO system activity was enhanced in rHuEpo–induced hypertension in rats with normal renal function and a resistance to NO was not developed in renal circulation. Further studies seem to be necessary to better clarify the exact mechanisms underlying the development of rHuEpo–induced hypertension.

Immunomodulatory activity of shikimic acid and quercitin in comparison with oseltamivir (Tamiflu) in an in vitro model.

The risk of an avian influenza pandemic has put oseltamivir (Tamiflu®) in the spotlight and has given rise to rumors that shikimic acid (SK), which is used for the synthesis of Tamiflu®, possesses therapeutic activity. This study was undertaken to determine whether SK, either alone or in combination with quercitin (QT) is able to modulate the release of IL‐6 and IL‐8 from peripheral blood mononuclear cells (PBMCs). The experiments were conducted comparing the properties of SK, both alone and in combination, with those of Tamiflu®. The incubation of PBMCs with 100 nM Tamiflu® or SK at two concentrations (10 nM; 100 nM) did not produce any change in IL‐6 and IL‐8 baseline levels (data expressed as incremental change vs. baseline). On the contrary, incubation with SK and QT at both concentrations (10 and 100 nM) produced a significant increase in the release of IL‐8 as compared to other groups (4.19 ± 0.82, SK‐QT 10 nM; 3.83 ± 1.17 SK‐QT 100 nM, P < 0.05 vs. baseline 1.00 ± 0.10, Tamiflu® 100 nM 1.35 ± 0.16, SK 10 nM 1.68 ± 0.15 and SK 100 nM 1.80 ± 0.48). The SK‐QT combination also proved to be effective in the upregulation of IL‐6 (3.08 ± 0.46, SK‐QT 10 nM; 3.60 ± 0.74 SK‐QT 100 nM, P < 0.05 vs. baseline 1.00 ± 0.26). According to these findings SK alone is not able to modulate innate immunity in antiviral terms. However, the data show that the SK + QT combination, even at low doses, may be effective for the modulation of innate immunity. J. Med. Virol. 80:741–745, 2008.

Repeated DNA elements in planarians of the Dugesia gonocephala group (Platyhelminthes, Tricladida)

The results of the isolation of repetitive DNA elements in the genome of Dugesia etrusca, a species of the Dugesia gonocephala group, are reported. These sequences, about 1.4 kb long, represent only a part of longer interspersed genomic units (De1 family) and appear to be limited to the genome of some planarians of this group, as indicated by a Southern blot analysis performed in different species and populations. The genomic relationships among different species and populations of the D. gonocephala group are discussed in relation to the results obtained in the present work.

Sucralfate modulates uPAR and EGFR expression in an experimental rat model of cervicitis.

Sucralfate is a drug used in the treatment of gastric and duodenal ulcer; it is cytoprotective and able to increase the bioavailability of several growth factors, modulating the wound healing process. In this study we tested the possible therapeutic effect of Sucralfate in the treatment of ulcerative lesion occurring in uterine cervix; to investigate such effect we used an experimental rat model of cervicitis in which the uPAR and EGFR expression were evaluated. Cervicitis was induced in wild and ovariectomized wistar female rats by an acetic acid-soaked tampon. The animals were divided into two main groups (4 and 7 days) and Sucralfate was administered topically until the day they were sacrificed. In order to distinguish physiological and drug-induced healing, quantitative and qualitative uPAR and EGFR expression were evaluated by using Western Blot and Immunohistochemistry techniques. Western Blot analysis demonstrated an increased expression of both receptors after 4 days from wounding in wild and ovariectomized animals. In particular in ovariectomized animals the expression of uPAR and EGFR increased after 4 days while it reduced following the administration of Sucralfate. In wild rats the same was observed for uPAR expression, while EGFR was different; in fact, its expression increased significantly at day 4 in the animals treated with the drug and only at day 7 in those untreated. Immunohistochemistry highlighted a noteworthy epithelial colocalization of EGFR and uPAR after 4 days in the animals treated with Sucralfate. We conclude that Sucralfate can promote the healing of ulcerative cervicitis and moreover, it reduces the normal healing time because of its modulatory property on uPAR and EGFR expression.

Molecular cytogenetics in planarians. I. Fluorescence in situ hybridization (FISH) of ribosomal DNA to mitotic chromosomes of Dugesia sicula Lepori (Turbellaria: Tricladida)

Summary The nucleolus organizing regions (NORs) were detected on mitotic chromosomes of the freshwater planarian Dugesia sicula Lepori, by the in situ fluorescence technique (FISH). The results show that ribosomal DNA (rDNA) sequences are located on medium-sized chromosomes, at telomeric positions. The application of the in situ hybridization method, which enables the molecular arrangement of specific DNAs to be visualized on the chromosomes, improves the karyotypic characterization in planarians and is of interest in a cytogenetic and cytotaxonomic perspective.