Dr. Alvarado-Livacic, Cristobal

Oxidative stress produces macromolecules dysfunction and cellular damage. Renal ischemia-reperfusion injury (IRI) induces oxidative stress, inflammation, epithelium and endothelium damage, and cessation of renal function. The IRI is an inevitable process during kidney transplantation. Preliminary studies suggest that aminoguanidine (AG) is an antioxidant compound. In this study, we investigated the antioxidant effects of AG (50 mg/kg, intraperitoneal) and its association with molecular pathways activated by IRI (30 min/48 h) in the kidney. The antioxidant effect of AG was studied measuring GSSH/GSSG ratio, GST activity, lipoperoxidation, iNOS, and Hsp27 levels. In addition, we examined the effect of AG on elements associated with cell survival, inflammation, endothelium, and mesenchymal transition during IRI. AG prevented lipid peroxidation, increased GSH levels, and recovered the GST activity impaired by IRI. AG was associated with inhibition of iNOS, Hsp27, endothelial activation (VE-cadherin, PECAM), mesenchymal markers (vimentin, fascin, and HSP47), and inflammation (IL-1β, IL-6, Foxp3, and IL-10) upregulation. In addition, AG reduced kidney injury (NGAL, clusterin, Arg-2, and TFG-β1) and improved kidney function (glomerular filtration rate) during IRI. In conclusion, we found new evidence of the antioxidant properties of AG as a renoprotective compound during IRI. Therefore, AG is a promising compound to treat the deleterious effect of renal IRI.

Background/Aims: Renal ischemia and reperfusion injury (IRI) involves oxidative stress, disruption of microvasculature due to endothelial cell damage, loss of epithelial cell polarity secondary to cytoskeletal alterations, inflammation, and the subsequent transition into a mesenchymal phenotype. Ischemic preconditioning (IPC) has been proposed as a therapeutic strategy to avoid/ameliorate the IRI. Since previous results showed that IPC could have differential effects in kidney cortex vs. kidney medulla, in the present study we analyzed the effectiveness and molecular mechanisms implicated in IPC in both kidney regions. Methods: We evaluated 3 experimental groups of BALB/c male mice: control (sham surgery); renal ischemia (30 min) by bilateral occlusion of the renal pedicle and reperfusion (48 hours) (I/R); and renal IPC (two cycles of 5 min of ischemia and 5 min of reperfusion) applied just before I/R. Acute kidney injury was evaluated by glomerular filtration rate (GFR), Neutrophil Gelatinase-Associated Lipocalin (NGAL) blood level, and histologic analysis. Oxidative stress was studied measurement the Glutathione S-Transferase (GST) activity, GSH/GSSG ratio, and lipoperoxidation levels. Inflammatory mediators (IL-1β, IL-6, Foxp3, and IL-10) were quantified by qRT-PCR. The endothelial (PECAM-1), epithelial (AQP-1), mesenchymal (Vimentin, Fascin, and Hsp47), iNOS, clusterin, and Hsp27 expression were evaluated (qRT-PCR and/or Western blot). Results: The IPC protocol prevented the decrease of GFR, reduced the plasma NGAL, and ameliorated morphological damage in the kidney cortex after I/R. The IPC also prevented the downregulation of GST activity, lipoperoxidation and ameliorated the oxidized glutathione. In addition, IPC prevented the upregulation of vimentin, fascin, and Hsp47, which was associated with the prevention of the downregulation of AQP1 after I/R. The protective effect of IPC was associated with the upregulation of Hsp27, Foxp3, and IL-10 expression in the renal cortex. However, the upregulation of iNOS, IL-1β, IL-6, and clusterin by I/R were not modified by IPC. Conclusion: IPC conferred better protection in the kidney cortex as compared to the kidney medulla. The protective effect of IPC was associated with amelioration of oxidative stress, tubular damage, and the induction of markers of Treg lymphocytes activity in the cortical region. Further studies are needed to evaluate if lower tubular cell stress/damage after I/R may explain the preferential induction of Treg response in the kidney cortex induced by IPC.

Objective This study investigated the role of oxidative damage and nitric oxide (NO) synthases in the fetal heart using a model of intrauterine growth restriction induced by uteroplacental circulation restriction (UCR). Methods New Zealand white rabbits kept under 12-h light cycles, with food and water provided ad libitum, were subjected at day 25 of pregnancy to 40–50% uteroplacental artery ligation. We analyzed the gene expression of enzymes linked to nitric oxide synthesis (iNOS, eNOS, HO-1, and ARG-2), hypoxia inducible factor 1 alpha (HIF-1α), and the state of oxidative stress (protein carbonyl levels) in fetal heart homogenates. Additionally, we studied the histological morphology of the fetal heart. Results We found that fetal growth restriction was associated with a significant reduction in heart weight but a normal heart/body weight ratio in UCR animals. Hematoxylin and eosin staining showed normal left and right ventricular thickness but increased vessel dilatation with hyperemia in the hearts of the UCR group. We observed HIF-1α, eNOS, p-eNOS, and iNOS induction concomitant with intensified protein carbonyl levels but observed no changes in HO-1 or ARG-2 expression, suggesting increased NO and oxidative stress in the hearts of UCR animals. Conclusion Uteroplacental circulation restriction increased NO-linked enzymes, oxidative damage, and dilated coronary vessels in fetal hearts.

On renal ischemia-reperfusion (I/R) injury, recruitment of neutrophils during the inflammatory process promotes local generation of oxygen and nitrogen reactive species, which, in turn, are likely to exacerbate tissue damage. The mechanism by which inducible nitric oxide synthase (iNOS) is involved in I/R has not been elucidated. In this work, the selective iNOS inhibitor l- N6-(1-iminoethyl)lysine (l-NIL) and the NOS substrate l-arginine were employed to understand the role of NOS activity on the expression of particular target genes and the oxidative stress elicited after a 30-min of bilateral renal ischemia, followed by 48-h reperfusion in Balb/c mice. The main findings of the present study were that pharmacological inhibition of iNOS with l-NIL during an I/R challenge of mice kidney decreased renal injury, prevented tissue loss of integrity, and improved renal function. Several novel findings regarding the molecular mechanism by which iNOS inhibition led to these protective effects are as follows: 1) a prevention of the I/R-related increase in expression of Toll-like receptor 4 (TLR-4), and its downstream target, IL-1β; 2) reduced oxidative stress following the I/R challenge; noteworthy, this study shows the first evidence of glutathione S-transferase (GST) inactivation following kidney I/R, a phenomenon fully prevented by iNOS inhibition; 3) increased expression of clusterin, a survival autophagy component; and 4) increased expression of nuclear factor of activated T cells 5 (NFAT-5) and its target gene aquaporin-1. In conclusion, prevention of renal damage following I/R by the pharmacological inhibition of iNOS with l-NIL was associated with the inactivation of proinflammatory pathway triggered by TLR-4, oxidative stress, renoprotection (autophagy inactivation), and NFAT-5 signaling pathway.