Endogenous Hepatocyte Growth Factor Attenuates Inflammatory Response in Glycerol-Induced Acute Kidney Injury

Background: Hepatocyte growth factor (HGF) is overexpressed after acute kidney injury (AKI). The aim of this study was to evaluate the role of endogenous HGF in the progression of the inflammatory response in glycerol-induced AKI (Gly-AKI) in rats. Methods: Renal and systemic HGF expressions were evaluated during the development of Gly-AKI. Subsequently, the blockade of endogenous HGF was analyzed in rats treated with anti-HGF antibody concomitant to glycerol injection. Apoptosis, cell infiltration and chemokine and cytokine profiles were investigated. Results: We detected an early peak of renal and plasma HGF protein expressions 3 h after glycerol injection. The pharmacological blockade of the endogenous HGF exacerbated the renal impairment, the tubular apoptosis, the renal expression of monocyte chemoattractant protein-1 and the macrophage, CD43+, CD4+ and CD8+ T lymphocytes renal infiltration. The analysis of mRNA expressions of Th1 (t-bet, TNF-alpha, IL-1 beta) and Th2 (gata-3, IL-4, IL-10) cytokines showed a Th1-polarized response in Gly-AKI rats that was aggravated with the anti-HGF treatment. Conclusion: Endogenous HGF attenuates the renal inflammatory response, leukocyte infiltration and Th1 polarization after glycerol injection. The control of cellular immune response may partly explain the protective effect of endogenous HGF in the development of Gly-AKI. Copyright (C) 2008 S. Karger AG, Basel

Bradykinin inducible receptor is essential to lipopolysaccharide-induced acute lung injury in mice

Lipopolysaccharides from gram-negative bacteria are amongst the most common causative agents of acute lung injury, which is characterized by an inflammatory response, with cellular infiltration and the release of mediators/cytokines. There is evidence that bradykinin plays a role in lung inflammation in asthma but in other types of lung inflammation its role is less clear. In the present study we evaluated the role of the bradykinin B(1) receptor in acute lung injury caused by lipopolysaccharide inhalation and the mechanisms behind bradykinin actions participating in the inflammatory response. We found that in C57BI/6 mice, the bradykinin B(1) receptor expression was up-regulated 24 h after lipopolysaccharide inhalation. At this time, the number of cells and protein concentration were significantly increased in the bronchoalveolar lavage fluid and the mice developed airway hyperreactivity to methacholine. In addition, there was an increased expression of tumor necrosis factor-alpha, interleukin-1 beta and interferon-gamma and chemokines (monocytes chemotactic protein-1 and KC) in the bronchoalveolar lavage fluid and in the lung tissue. We then treated the mice with a bradykinin B, receptor antagonist, R-954 (Ac-Orn-[Oic(2), alpha-MePhe(5), D-beta Nal(7), Ile(8)]desArg(9)-bradykinin), 30 min after lipopolysaccharide administration. We observed that this treatment prevented the airway hyperreactivity as well as the increased cellular infiltration and protein content in the bronchoalveolar lavage fluid. Moreover, R-954 inhibited the expression of cytokines/chemokines. These results implicate bradykinin, acting through B(1) receptor, in the development of acute lung injury caused by lipopolysaccharide inhalation. (C) 2010 Elsevier B.V. All rights reserved.

Potent Cardioprotective Effect of the 4-Anilinoquinazoline Derivative PD153035: Involvement of Mitochondrial K(ATP) Channel Activation

Background: The aim of the present study was to evaluate the protective effects of the 4-anilinoquinazoline derivative PD153035 on cardiac ischemia/reperfusion and mitochondrial function. Methodology/Principal Findings: Perfused rat hearts and cardiac HL-1 cells were used to determine cardioprotective effects of PD153035. Isolated rat heart mitochondria were studied to uncover mechanisms of cardioprotection. Nanomolar doses of PD153035 strongly protect against heart and cardiomyocyte damage induced by ischemia/reperfusion and cyanide/aglycemia. PD153035 did not alter oxidative phosphorylation, nor directly prevent Ca(2+) induced mitochondrial membrane permeability transition. The protective effect of PD153035 on HL-1 cells was also independent of AKT phosphorylation state. Interestingly, PD153035 activated K(+) transport in isolated mitochondria, in a manner prevented by ATP and 5-hydroxydecanoate, inhibitors of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)). 5-Hydroxydecanoate also inhibited the cardioprotective effect of PD153035 in cardiac HL-1 cells, demonstrating that this protection is dependent on mitoK(ATP) activation. Conclusions/Significance: We conclude that PD153035 is a potent cardioprotective compound and acts in a mechanism involving mitoK(ATP) activation.

Antioxidant treatment reduces matrix metalloproteinase-2-induced vascular changes in renovascular hypertension

Mounting evidence indicates that structural and functional vascular changes associated with two-kidney, one-clip (2K-1C) hypertension result, at least in part, from altered activity of matrix metalloproteinases (MMPs). Because MMPs are upregulated by increased formation of reactive oxygen species (ROS), we hypothesized that antioxidant approaches could attenuate the increases in MMP-2 expression/activity and the vascular dysfunction and remodeling associated with 2K-1C hypertension. Sham-operated or 2K-1C hypertensive rats were treated with tempol 18 mg/kg/day or apocyanin 25 mg/kg/day (or vehicle). Systolic blood pressure was monitored weekly. After 8 weeks of treatment, aortic rings were isolated to assess endothelium-dependent and -independent relaxation. Quantitative morphometry of structural changes in the aortic wall was studied in hematoxylin/eosin sections. Aortic and systemic ROS levels were measured using dihydroethidine and thiobarbituric acid-reactive substances, respectively. Aortic MMP-2 levels and activity were determined by gelatin and in situ zymography, fluorimetry, and immunohistochemistry. Tempol and apocyanin attenuated 2K-1C hypertension (181 +/- 20.8 and 192 +/- 17.6 mm Hg, respectively, versus 213 +/- 18 mm Hg in hypertensive controls; both p<0.05) and prevented the reduction in endothelium-dependent vasorelaxation found in 2K-1C rats. Tempol, but not apocyanin (p>0.05), prevented the vascular remodeling found in 2K-1C rats (all p<0.01). Tempol was more effective than apocyanin in attenuating hypertension-induced increases in oxidative stress (both p<0.05), MMP-2 levels, and MMP-2 activity in hypertensive rats (all p<0.05). Our results suggest that antioxidant approaches decrease MMP-2 upregulation and attenuate the vascular dysfunction and remodeling during 2K-1C hypertension. (C) 2009 Elsevier Inc. All rights reserved.

Inflammatory Cytokines during Liver Transplantation: Prospective Randomized Trial Comparing Conventional and Piggyback Techniques

Background/Aims: Cytokines have a significant role in the response to injury following liver transplantation, but the origin and course of such molecules are not completely known. The aim of this study was to evaluate the production and liver metabolism of the inflammatory cytokines interleukin (IL)-1 beta, IL-6, IL-8, interferon (IFN)-Y and tumor necrosis factor (TNF)-alpha in orthotopic liver transplantation (OLT), comparing the conventional and the piggyback methods. Methodology: We performed a study of 30 patients who underwent elective OLT and were randomized for the conventional or piggyback techniques at the beginning of the operation. The amount of cytokines and their hepatic metabolism were calculated based on plasma concentrations and vascular blood flow at 2, 5, 10, 15, 30, 60, 90, and 120 minutes after revascularization. Results: The amount of IL-1 beta in portal blood was higher in patients who underwent surgery using the conventional technique (estimate interest = 63,783.9 +/- 16,586.1 pg/min, versus 11,979.6 +/- 16,585.7 pg/min in the piggyback group, p=0.035). There were no significant differences between the two operative`s methods for IL-6, IL-8, IFN-Y and TNF-alpha production. The hepatic metabolism of cytokines was not different between groups. Although all the curves showed higher amounts of cytokines with the conventional technique, these were not statistically significant. Conclusion: The study shows the similarity between the two techniques concerning the stimuli for the production of inflammatory molecules.

Effect of IIb-IIIa Glycoprotein Inhibitors in a Partial Limb Amputation Model Submitted to Warm Ischemia in Rats

Viability and functional results of a segment replantation depend on the prevention of deleterious effects of ischemia. Prolonged ischemia leads to alterations in the microcirculation: thrombosis, edema, production of oxygen free radicals, and platelet aggregation. The effect of IIb-IIIa glycoprotein inhibitors was tested in a partial limb amputation model submitted to warm ischemia. The male Wistar rats were divided into four groups: G1 with 0 hours of ischemia and saline (n = 20), G2 with 6 hours of ischemia and saline (n = 24), G3 with 6 hours of ischemia and abciximab (n = 23), and G4 with 6 hours of ischemia and tirofiban (n = 29). The limbs were observed for 7 days and classified as viable or nonviable. Viability, and mortality rates were obtained and analyzed by Q-square and Fisher exact tests (p < 0.05). The viability rates were 100% (G1), 30% (G2), 77.78% (G3), and 80.95% (G4). G2 was statistically different from G1, G3, and G4. G1, G3, and G4 were not statistically different. Transoperative and postoperative mortalities were not statistically different. The administration of abciximab and tirofiban improved limb salvage after ischemia and reperfusion and did not modify mortality rates significantly.

Comparison Between Perfadex and Locally Manufactured Low-Potassium Dextran Solution for Pulmonary Preservation in an Ex Vivo Isolated Lung Perfusion Model

Introduction. Lung tranplantation, a consolidated treatment for end-stage lung disease, utilizes preservation solutions, such as low potassium dextran (LPD), to mitigate ischemia reperfusion injury. We sought the local development of LPD solutions in an attempt to facilitate access and enhance usage. We also sought to evaluate the effectiveness of a locally manufactured LPD solution in a rat model of ex vivo lung perfusion. Methods. We randomized the following groups \?\adult of male Wistar rats (n = 25 each): Perfadex (LPD; Vitro life, Sweden); locally manufactured LPD-glucose (LPDnac) (Farmoterapica, Brazil), and normal saline solution (SAL) with 3 ischemic times (6, 12, and 24 hours). The harvested heart lung blocks were flushed with solution at 4 C. After storage, the blocks were connected to an IL-2 Isolated Perfused Rat or Guinea Pig Lung System (Harvard Apparatus) and reperfused with homologous blood for 60 minutes. Respiratory mechanics, pulmonary artery pressure, perfusate blood gas analysis, and lung weight were measured at 10-minute intervals. Comparisons between groups and among ischemic times were performed using analysis of variance with a 5% level of significance. Results. Lungs preserved for 24 hours were nonviable and therefore excluded from the analysis. Those preserved for 6 hours showed better ventilatory mechanics when compared with 12 hours. The oxygenation capacity was not different between lungs flushed with LPD or LPDnac, regardless of the ischemic time. SAL lungs showed higher PCO(2) values than the other solutions. Lung weight increased over time during perfusion; however, there were no significant differences among the tested solutions (LPD, P = .23; LPDnac, P = .41; SAL, P = .26). We concluded that the LPDnac solution results in gas exchange were comparable to the original LPD (Perfadex); however ventilatory mechanics and edema formation were better with LPD, particularly among lungs undergoing 6 hours of cold ischemia.

Microcirculatory effects of local and remote ischemic preconditioning in supraceliac aortic clamping

Introduction: Supraceliac aortic clamping in major vascular procedures promotes splanchnic ischemia and reperfusion (I/R) injury that may induce endothelial dysfunction, widespread inflammation, multiorgan dysfunction, and death. We tested the hypothesis that local or remote ischemic preconditioning (IPC) may be protective against injury after supraceliac aortic clamping through the modulation of mesenteric leukocyte-endothelial interactions, as evaluated with intravital microscopy and expression of adhesion molecules. Methods: Fifty-six male Wistar rats (weight, 190 to 250 g), were divided into four groups of 14 rats each: control sham surgery without aortic occlusion; I/R through supraceliac aortic occlusion for 20 minutes, followed by 120 minutes of reperfusion; local IPC through supraceliac aortic occlusion for two cycles of 5 minutes of ischemia and 5 minutes of reperfusion, followed by the same protocol of the IR group; remote IPC through infrarenal aortic occlusion for two cycles of 10 minutes of ischemia and 10 minutes of reperfusion, followed by the same protocol of the IR group. Seven animals per group were used to evaluate in vivo leukocyte-endothelial interactions in postcapillary venules with intravital microscopy and another seven animals per group were used to collect mesentery samples for inmmnohistochemistry demonstration of adhesion molecules expression. Results: Supraceliac aortic occlusion increased the number of rolling leukocytes with slower velocities and increased the number of adherent leukocytes to the venular surface and leukocyte migration to the interstitium. The expression of P-selectin, E-selectin, and intercellular adhesion molecule-1 was also increased significantly after I/R. Local or remote IPC reduced the leukocyte recruitment in vivo and normalized the expression of adhesion molecules. Conclusions: Local or remote IPC reduces endothelial dysfunction on mesenteric microcirculation caused by I/R injury after supraceliac aortic clamping. (J Vase Surg 2010;52:1321-9.) Clinical Relevance: The present study demonstrates that ischemia and reperfusion injury induced by supraceliac aortic occlusion promotes endothelial dysfunction and leukocyte recruitment on mesenteric microcirculation. Local and remote preconditioning reduced leukocyte-endothelial interactions and normalized the expression of endothelial adhesion molecules involved in this process. Although we recognize the limitation of an experimental model, our findings suggest that local and remote ischemic preconditioning minimize the endothelial dysfunction and leukocyte recruitment events that play a central role in systemic inflammation and multiorgan dysfunction after major aortic reconstructions.

Endoscopic features in a model of multivisceral xenotransplantation

Introduction: Xenotransplantation and multivisceral transplantation are advanced therapeutic methods that still require a scientific basis. There are no experimental models of multivisceral transplantation available, particularly not the monitoring by endoscopy. Here, we describe the endoscopic features in a model of multivisceral xenotransplantation. Methods: The distal esophagus, stomach, intestine, colon, liver, pancreas, and the kidneys with a common vascular pedicle were harvested from rabbits and implanted in swine (group I, n = 9) or in rabbits (group II, n = 4). Endoscopy was performed in the stomach, jejunum, and ascending colon at four consecutive time points (immediate after surgery and 10, 90, and 180 min after reperfusion). Lesions were macroscopically graded as mild, moderate, and severe. Biopsies were taken following sacrifice at 180 min after reperfusion. Results: In group I, the stomach, jejunum, and colon manifested a progression of lesions with predominance of mild lesions after 10 min, mild to moderate lesions after 90 min, and moderate to severe lesions after 180 min. In animals from group II, endoscopy showed normal features at all time points after reperfusion. Histopathologic analysis confirmed the diagnosis of hyperacute rejection in group I. Grafts from group II animals presented normal or mild ischemic/reperfusion injury. Conclusion: All animals subjected to multivisceral xenotransplantation showed a progression of endoscopic lesions with time after transplantation, while animals subjected to allotransplantation showed no aberrations in endoscopy. We conclude that endoscopy is a useful tool in the assessment of hyperacute rejection of a xenograft.

Experimental multivisceral xenotransplantation

Background: Organ shortage impairs the proposition of multivisceral transplantation to treat multiple organ failure. Interspecies (xeno) transplantation is a valid solution for organ shortage; however, suitable models of this advance are lacking. We describe an effective model of multivisceral xenotransplantation to study hyperacute rejection. Methods: Under general anesthesia, we in block recovered the distal esophagus, stomach, small bowel, colon, liver, pancreas, spleen, and kidneys from donors and implanted heterotopically in the lower abdomen of recipients. Animals were divided into four groups: I-canine donor, swine recipient (n = 6); II - swine donor, canine recipient (n = 5); III-canine donor, canine recipient (n = 4); and IV-swine donor, swine recipient (n = 5). Groups I and 11 comprised experimental (xenotransplantation) and III and IV control groups (allotransplantation). During the experiment, we appraised recipient evolution and graft modification by sequential biopsy up to 3 h. At this time, we killed animals for autopsy (experimental end point). Results: We accomplished all experiments successfully. Every grafts attained customary appearance and convenient urine output immediately after unclamp. Around 15 min after reperfusion, xenografts achieved signs of progressive hyperacute rejection and absence of urine output. At the end of experiments we observed moderate to severe hyperacute rejection at small bowel, colon, mesenteric lymph node, liver, spleen, pancreas, and kidney, while stomach and esophagus achieved mild lesions. In contrast, allograft achieved normal or minimum ischemia/reperfusion injury and constant urine output. Conclusion: The present procedure assembles a simple and effective model to study multivisceral xenotransplantation and may ultimately spread researches toward hyperacute rejection.

EFFECTS OF A POTENT PEROXYNITRITE DECOMPOSITION CATALYST IN MURINE MODELS OF ENDOTOXEMIA AND SEPSIS

Excessive free-radical production due to various bacterial components released during bacterial infection has been linked to cell death and tissue injury. Peroxynitrite is a highly reactive oxidant produced by the combination of nitric oxide (NO) and superoxide anion, which has been implicated in cell death and tissue injury in various forms of critical illness. Pharmacological decomposition of peroxynitrite may represent a potential therapeutic approach in diseases associated with the overproduction of NO and superoxide. In the present study, we tested the effect of a potent peroxynitrite decomposition catalyst in murine models of endotoxemia and sepsis. Mice were injected i.p. with LPS 40 mg/kg with or without FP15 [Fe(III) tetrakis-2-(N-triethylene glycol monomethyl ether) pyridyl porphyrin] (0.1, 0.3, 1, 3, or 10 mg/kg per hour). Mice were killed 12 h later, followed by the harvesting of samples from the lung, liver, and gut for malondialdehyde and myeloperoxidase measurements. In other subsets of animals, blood samples were obtained by cardiac puncture at 1.5, 4, and 8 h after LPS administration for cytokine (TNF-alpha, IL-1 beta, and IL-10), nitrite/nitrate, alanine aminotransferase, and blood urea nitrogen measurements. Endotoxemic animals showed an increase in survival from 25% to 80% at the FP15 doses of 0.3 and 1 mg/kg per hour. The same dose of FP15 had no effect on plasma levels of nitrite/nitrate. There was a reduction in liver and lung malondialdehyde in the endotoxemic animals pretreated with FP15, as well as in hepatic myeloperoxidase and biochemical markers of liver and kidney damage (alanine aminotransferase and blood urea nitrogen). In a bacterial model of sepsis induced by cecal ligation and puncture, FP15 treatment (0.3 mg/kg per day) significantly protected against mortality. The current data support the view that peroxynitrite is a critical factor mediating liver, gut, and lung injury in endotoxemia and septic shock: its pharmacological neutralization may be of therapeutic benefit.

Protective Effect of N-acetylcysteine on Early Outcomes of Deceased Renal Transplantation

We investigated the effects of the antioxidant N-acetylcysteine (NAC) on early outcomes of deceased donor renal transplantation. Between April 2005 and June 2008, adult primary graft recipients of deceased renal donors were assigned to treatment (n = 38) or control (n = 36) groups and evaluated for 90 days and one year after renal transplantation. The treatment group received NAC orally (600 mg twice daily) from day 0 to 7 postoperatively. Renal function was determined by serum creatinine, MDRD and Cockcroft-Gault estimated GFR (eGFR), delayed graft function (DGF) and dialysis free Kaplan-Meier estimate curve. Serum levels of thiobarbituric acid reactive substances (TBARS), were employed as markers of oxidative stress. The NAC group displayed a lower mean serum creatinine during the first 90 days (P = .026) and at 1 year after transplantation (P = .005). Furthermore, the NAC group showed a higher mean eGFR throughout the first 90 days and at 1 year. DGF was lower among the NAC group (P = .017) and these recipients required fewer days of dialysis (P = .012). Oxidative stress was significantly attenuated with NAC (P < .001). Our results suggested that NAC enhanced early outcomes of deceased donor renal transplantation by attenuating oxidative stress.

Comparison of Two Models for Evaluation Histopathology of Experimental Renal Ischemia

Renal ischemia/reperfusion (I/R) injury is one of the frequent causes of acute renal failure (ARF) due to the complex, interrelated sequence of events, that result in damage to and death of kidney cells. Cells of the proximal tubular epithelium are especially susceptible to I/R injury, leading to acute tubular necrosis, which plays a pivotal role in the pathogenesis of ARE Several models have been explicated to assess morphological changes, including those of Jabonski et al. and Goujon et al. We compared the 2 models for histopathological evaluation of 30- or 120-minute periods of renal ischemia followed by 24-hour reperfusion in rats. Several changes were observed after application of the 2 models: proximal tubular cell necrosis, loss of brush border, vacuolization, denudation of tubular basement membrane as a consequence of flattening of basal cells, and presence of intratubular exfoliated cells in the lumen of proximal convoluted tubules at various stages of degeneration (karyorexis, kariopyknosis and karyolysis). Evaluating tubular lesions after 2 periods of experimental ischemia with light microscopy allowed us to conclude that the Goujon classification better characterized the main changes in cortical renal tubules after ischemia.

Asymmetric dimethylarginine endogenous inhibition of nitric oxide synthase causes differential vasculature effects

Background: Asymmetric dimethylarginine (ADMA), produced during protein metabolism, is an endogenous inhibitor of nitric oxide synthase, but little is known about its direct vasoactive properties in different arterial beds. Material/Methods: Segments of canine coronary, renal, and femoral arteries were pretreated with increasing concentrations of ADMA, and endothelial function was evaluated in organ chambers. Results: In precontracted canine coronary arteries, the highest concentrations of ADMA inhibited endothelium-dependent relaxation mediated by acetylcholine (n=7), but no concentration of ADMA inhibited receptor-independent relaxation mediated by calcium ionophore (n=7) (P<.001). The effect of ADMA on acetylcholine-mediated relaxation was shown to be competitive inhibition of the nitric oxide synthase pathway, because the addition of L-arginine (10(-3) M), but not D-arginine (101 M), reversed the effect produced by 10(-5) M ADMA. Further, ADMA did not alter endothelium-independent relaxation mediated by sodium nitroprusside (10(-9) to 10(-6) M; n=7). Femoral arteries (n=7) and renal arteries (n=7) were more sensitive to ADMA than were coronary arteries, and they demonstrated significant ADMA inhibition to receptor dependent relaxation induced by acetylcholine (P=.03 and P=.01, respectively) and to receptor-independent relaxation induced by calcium ionophore (P=.02 and P=.01, respectively). Conclusions: Endothelium-dependent relaxation mediated by ADMA is more marked in femoral and renal arteries than in coronary arteries. The response in coronary arteries may be overall protective. Considering these different effects in various artery types, the role of ADMA as a confiable and specific cardiovascular risk factor is questioned.

Increased activities of cardiac matrix metalloproteinases matrix metalloproteinase (MMP)-2 and MMP-9 are associated with mortality during the acute phase of experimental Trypanosoma cruzi infection

The strong inflammatory reaction that occurs in the heart during the acute phase of Trypanosoma cruzi infection is modulated by cytokines and chemokines produced by leukocytes and cardiomyocytes. Matrix metalloproteinases (MMPs) have recently emerged as modulators of cardiovascular inflammation. In the present study we investigated the role of MMP-2 and MMP-9 in T. cruzi-induced myocarditis, by use of immunohistochemical analysis, gelatin zymography, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction to analyze the cardiac tissues of T. cruzi-infected C57BL/6 mice. Increased transcripts levels, immunoreactivity, and enzymatic activity for MMP-2 and MMP-9 were observed by day 14 after infection. Mice treated with an MMP inhibitor showed significantly decreased heart inflammation, delayed peak in parasitemia, and improved survival rates, compared with the control group. Reduced levels of cardiac tumor necrosis factor-alpha, interferon-gamma, serum nitrite, and serum nitrate were also observed in the treated group. These results suggest an important role for MMPs in the induction of T. cruzi-induced acute myocarditis.