Clinical Outcomes and Genetic Expression Profile in Human Liver Graft Dysfunction During Ischemia/Reperfusion Injury

Introduction. This study aims to compare the molecular gene expression during ischemia reperfusion injury. Several surgical times were considered: in the beginning of the harvesting (T0), at the end of the cold ischemia period (T1), and after reperfusion (T2) and compared with graft dysfunction after liver transplant (OLT). Methods. We studied 54 patients undergoing OLT. Clinical, laboratory data, and histologic data (Suzuki classification) as well as the Survival Outcomes Following Liver Transplantation (SOFT) score were used and compared with the molecular gene expression of the following genes: Interleukin (IL)-1b, IL-6, tumor necrosis factor-a, perforin, E-selectin (SELE), Fas-ligand, granzyme B, heme oxygenase-1, and nitric oxide synthetase. Results. Fifteen patients presented with graft dysfunction according to SOFT criteria. No relevant data were obtained by comparing the variables graft dysfunction and histologic variables. We observed a statistically significant relation between SELE at T0 (P ¼ .013) and IL-1b at T0 (P ¼ .028) and early graft dysfunction. Conclusions. We conclude that several genetically determined proinflammatory expressions may play a critical role in the development of graft dysfunction after OLT.

Identification of mechanisms controlling the transcriptional activity of nuclear factor kappa B (NF-κB) p65/RelA

Dissertação para a obtenção do grau de doutor em Biologia pelo Instituto de Tecnologia Química e Biológica. Universidade Nova de Lisboa.

Convergencia de vías de señalización en un modelo de apoptosis

La sepsis es un evento inflamatorio generalizado del organismo inducido por un daño causado generalmente por un agente infeccioso. El patógeno más frecuentemente asociado con esta entidad es el Staphylococcus aureus, responsable de la inducción de apoptosis en células endoteliales debida a la producción de ceramida. Se ha descrito el efecto protector de la proteína C activada (PCA) en sepsis y su relación con la disminución de la apoptosis de las células endoteliales. En este trabajo se analizó la activación de las quinasas AKT, ASK1, SAPK/JNK y p38 en un modelo de apoptosis endotelial usando las técnicas de Western Blotting y ELISA. Las células endoteliales (EA.hy926), se trataron con C2-ceramida (130μM) en presencia de inhibidores químicos de cada una de estas quinasas y PCA. La supervivencia de las células en presencia de inhibidores químicos y PCA fue evaluada por medio de ensayos de activación de las caspasas 3, 7 y 9, que verificaban la muerte celular por apoptosis. Los resultados evidencian que la ceramida reduce la activación de AKT y aumenta la activación de las quinasas ASK, SAPK/JNK y p38, en tanto que PCA ejerce el efecto contrario. Adicionalmente se encontró que la tiorredoxina incrementa la activación/fosforilación de AKT, mientras que la quinasa p38 induce la defosforilación de AKT.

Ascorbate does not protect macrophages against apoptosis induced by oxidised low density lipoprotein

Apoptosis of macrophages and smooth muscle cells is observed in atherosclerotic lesions and may play an important role in the disease progression. Oxidised low density lipoprotein (LDL) is cytotoxic and induces apoptosis in a variety of cell types. We reported previously that ascorbate protects arterial smooth muscle cells from apoptosis induced by oxidised LDL containing the peak levels of lipid hydroperoxides. We now demonstrate that macrophages undergo apoptosis when treated with this species of oxidised LDL, as detected by increased annexin V binding and DNA fragmentation. Ascorbate treatment of macrophages did not protect against the cytotoxicity of oxidised LDL, and modestly increased the levels of annexin V binding and DNA fragmentation. Oxidised LDL treatment also increased the expression of the antioxidant stress protein heme oxygenase-1 in macrophages; however, this increase was markedly attenuated by ascorbate pretreatment. Although apoptosis induced by oxidised LDL was modestly promoted by ascorbate, ascorbate apparently decreased the levels of oxidative stress in macrophages, suggesting that this pro-apoptotic effect was not mediated by a pro-oxidant mechanism, but may instead have been due to intracellular protection of the apoptotic machinery by ascorbate. (c) 2006 Elsevier Inc. All rights reserved.

Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages - Activation by oxidatively modified LDL and 4-hydroxynonenal

CD36 is an important scavenger receptor mediating uptake of oxidized low- density lipoproteins ( oxLDLs) and plays a key role in foam cell formation and the pathogenesis of atherosclerosis. We report the first evidence that the transcription factor Nrf2 is expressed in vascular smooth muscle cells, and demonstrate that oxLDLs cause nuclear accumulation of Nrf2 in murine macrophages, resulting in the activation of genes encoding CD36 and the stress proteins A170, heme oxygenase- 1 ( HO- 1), and peroxiredoxin I ( Prx I). 4- Hydroxy- 2- nonenal ( HNE), derived from lipid peroxidation, was one of the most effective activators of Nrf2. Using Nrf2- deficient macrophages, we established that Nrf2 partially regulates CD36 expression in response to oxLDLs, HNE, or the electrophilic agent diethylmaleate. In murine aortic smooth muscle cells, expressing negligible levels of CD36, both moderately and highly oxidized LDL caused only limited Nrf2 translocation and negligible increases in A170, HO- 1, and Prx I expression. However, treatment of smooth muscle cells with HNE significantly enhanced nuclear accumulation of Nrf2 and increased A170, HO- 1, and Prx I protein levels. Because PPAR-gamma can be activated by oxLDLs and controls expression of CD36 in macrophages, our results implicate Nrf2 as a second important transcription factor involved in the induction of the scavenger receptor CD36 and antioxidant stress genes in atherosclerosis.

Carbon monoxide protects against oxidant-induced apoptosis via inhibition of Kv2.1

Oxidative stress induces neuronal apoptosis and is implicated in cerebral ischemia, head trauma, and age-related neurodegenerative diseases. An early step in this process is the loss of intracellular K(+) via K(+) channels, and evidence indicates that K(v)2.1 is of particular importance in this regard, being rapidly inserted into the plasma membrane in response to apoptotic stimuli. An additional feature of neuronal oxidative stress is the up-regulation of the inducible enzyme heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe(2+), and carbon monoxide (CO). CO provides neuronal protection against stresses such as stroke and excitotoxicity, although the underlying mechanisms are not yet elucidated. Here, we demonstrate that CO reversibly inhibits K(v)2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of K(v)2.1 in HEK293 cells increases their vulnerability to oxidant-induced apoptosis, and this is reversed by CO. In hippocampal neurons, CO selectively inhibits K(v)2.1, reverses the dramatic oxidant-induced increase in K(+) current density, and provides marked protection against oxidant-induced apoptosis. Our results provide a novel mechanism to account for the neuroprotective effects of CO against oxidative apoptosis, which has potential for therapeutic exploitation to provide neuronal protection in situations of oxidative stress.

Carbon monoxide inhibits L-type Ca2+ channels via redox modulation of key cysteine residues by mitochondrial reactive oxygen species

Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca2+ currents and the recombinant alpha1C subunit of the human cardiac L-type Ca2+ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca2+ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channel's cytoplasmic C-terminal tail, resulting in channel inhibition.

Carbon monoxide inhibition of Cav3.2 T-type Ca2+ channels reveals tonic modulation by thioredoxin

T-type Ca2+ channels play diverse roles in tissues such as sensory neurons, vascular smooth muscle, and cancers, where increased expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1) is often found. Here, we report regulation of T-type Ca2+ channels by carbon monoxide (CO) a HO-1 by-product. CO (applied as CORM-2) caused a concentration-dependent, poorly reversible inhibition of all T-type channel isoforms (Cav3.1-3.3, IC50 ∼3 μM) expressed in HEK293 cells, and native T-type channels in NG108-15 cells and primary rat sensory neurons. No recognized CO-sensitive signaling pathway could account for the CO inhibition of Cav3.2. Instead, CO sensitivity was mediated by an extracellular redox-sensitive site, which was also highly sensitive to thioredoxin (Trx). Trx depletion (using auranofin, 2-5 μM) reduced Cav3.2 currents and their CO sensitivity by >50% but increased sensitivity to dithiothreitol ∼3-fold. By contrast, Cav3.1 and Cav3.3 channels, and their sensitivity to CO, were unaffected in identical experiments. Our data propose a novel signaling pathway in which Trx acts as a tonic, endogenous regulator of Cav3.2 channels, while HO-1-derived CO disrupts this regulation, causing channel inhibition. CO modulation of T-type channels has widespread implications for diverse physiological and pathophysiological mechanisms, such as excitability, contractility, and proliferation

Changes in gene expression induced by H(2)O(2) in cardiac myocytes.

Oxidative stress induces cardiac myocyte apoptosis. At least some effects are probably mediated through changes in gene expression. Using Affymetrix arrays, we examined the changes in gene expression induced by H(2)O(2) (0.04, 0.1, and 0.2mM; 2 and 4h) in rat neonatal ventricular myocytes. Changes in selected upregulated genes were confirmed by ratiometric RT-PCR. p21(Cip1/Waf1) was one of the only two genes upregulated in all conditions studied. Of the heat shock proteins, only Hsp70/70.1 was induced by H(2)O(2) with no change in the expression of Hsp25, Hsp60 or Hsp90. Heme oxygenase 1 was also potently upregulated, but not heme oxygenases 2 or 3. Of the intercellular adhesion proteins, syndecan-1 was significantly upregulated in response to H(2)O(2), with little change in the expression of other syndecans and no change in expression of any of the integrins studied. Thus, oxidative stress, exemplified by H(2)O(2), selectively promotes the expression of specific gene family members.

Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

Modulation of inflammatory response by selective inhibition of cyclooxygenase-1 and cyclooxygenase-2 in acute kidney injury

This work explored the role of inhibition of cyclooxygenases (COXs) in modulating the inflammatory response triggered by acute kidney injury. C57Bl/6 mice were used. Animals were treated or not with indomethacin (IMT) prior to injury (days -1 and 0). Animals were subjected to 45 min of renal pedicle occlusion and sacrificed at 24 h after reperfusion. Serum creatinine and blood urea nitrogen, reactive oxygen species (ROS), kidney myeloperoxidase (MPO) activity, and prostaglandin E2 (PGE(2)) levels were analyzed. Tumor necrosis factor (TNF)-alpha, t-bet, interleukin (IL)-10, IL-1 beta, heme oxygenase (HO)-1, and prostaglandin E synthase (PGES) messenger RNA (mRNA) were studied. Cytokines were quantified in serum. IMT-treated animals presented better renal function with less acute tubular necrosis and reduced ROS and MPO production. Moreover, the treatment was associated with lower expression of TNF-alpha, PGE(2), PGES, and t-bet and upregulation of HO-1 and IL-10. This profile was mirrored in serum, where inhibition of COXs significantly decreased interferon (IFN)-gamma, TNF-alpha, and IL-12 p70 and upregulated IL-10. COXs seem to play an important role in renal ischemia and reperfusion injury, involving the secretion of pro-inflammatory cytokines, activation of neutrophils, and ROS production. Inhibition of COX pathway is intrinsically involved with cytoprotection.

Mesenchymal Stem Cells Attenuate Renal Fibrosis Through Immune Modulation and Remodeling Properties in a Rat Remnant Kidney Model

Mesenchymal stem cells (MSCs) have regenerative properties in acute kidney injury, but their role in chronic kidney diseases is still unknown. More specifically, it is not known whether MSCs halt fibrosis. The purpose of this work was to investigate the role of MSCs in fibrogenesis using a model of chronic renal failure. MSCs were obtained from the tibias and femurs of male Wistar-EPM rats. Female Wistar rats were subjected to the remnant model, and 2 vertical bar x vertical bar 10(5) MSCs were intravenously administrated to each rat every other week for 8 weeks or only once and followed for 12 weeks. SRY gene expression was observed in female rats treated with male MSCs, and immune localization of CD73(+)CD90(+) cells at 8 weeks was also assessed. Serum and urine analyses showed an amelioration of functional parameters in MSC-treated animals at 8 weeks, but not at 12 weeks. Masson`s trichrome and Sirius red staining demonstrated reduced levels of fibrosis in MSC-treated animals. These results were corroborated by reduced vimentin, type I collagen, transforming growth factor beta, fibroblast specific protein 1 (FSP-1), monocyte chemoattractant protein 1, and Smad3 mRNA expression and alpha smooth muscle actin and FSP-1 protein expression. Renal interleukin (IL)-6 and tumor necrosis factor alpha mRNA expression levels were significantly decreased after MSC treatment, whereas IL-4 and IL-10 expression levels were increased. All serum cytokine expression levels were decreased in MSC-treated animals. Taken together, these results suggested that MSC therapy can indeed modulate the inflammatory response that follows the initial phase of a chronic renal injury. The immunosuppressive and remodeling properties of MSCs may be involved in the decreased fibrosis in the kidney. STEM CELLS 2009;27:3063-3073

Critical involvement of Th1-related cytokines in renal injuries induced by ischemia and reperfusion

Renal ischemia and reperfusion injury (IRI) is considered an inflammatory syndrome. To move forward in its pathogenesis, we exploited the role of several cytokines on renal damages triggered by IRI. Specifically to evaluate the role of Th1 immune profile in this system, IL-12, IFN-gamma, and IFN-gamma/IL-12 deficient (KO) mice on C57BL/6 background and their controls were subjected to IRI. In each group, blood and kidney samples were harvested. Renal function was evaluated by serum creatinine and renal morphometric analyses. Gene expression of IL-6 and HO-1 were also investigated by Q-PCR. IFN-gamma KO animals presented the highest impairment in renal function compared to controls. Conversely, IL-12 KO animals were absolutely protected and, in a lesser extent, IFN-gamma/IL-12 KO double knockout was also protected from IRI. Gene expression analyses showed higher expression of HO-1, a cytoprotective gene, and IL-6, a pro-inflammatory cytokine, in IFN-gamma deficient animals subjected to IRI. Our results confirm that Th1 related cytokines such as IL-12 and IFN-gamma are critically involved in renal ischemia and reperfusion injury. (C) 2008 Elsevier B.V. All rights reserved.

A role for regulatory T cells in renal acute kidney injury

Ischemia reperfusion injury (IRI) is a potential contributor for the development of chronic allograft nephropathy. T cells are important mediators of injury, even in the absence of alloantigens. We performed a depletion of TCD4(+)CTLA4(+)Foxp3(+) cells with anti-CD25(PC61), a treatment with anti-GITR (DTA-1) and rat-IgG, followed by 45 min of ischemia and 24/72 h of reperfusion, and then analyzed blood urea, kidney histopathology and gene expression in kidneys by QReal Time PCR. After 24 h of reperfusion, depletion of TCD4(+)CTLA4(+)Foxp3(+) cells reached 30.3%(spleen) and 67.8%(lymph nodes). 72 h after reperfusion depletion reached 43.1%(spleen) and 90.22%(lymph nodes) and depleted animals presented with significantly poorer renal function, while DTA-1 (anti-GITR)-treated ones showed a significant protection, all compared to serum urea from control group (IgG: 150.10 +/- 50.04; PC61: 187.23 +/- 31.38; DTA-1: 64.53 +/- 25.65, mg/dL, p<0.05). These data were corroborated by histopathology. We observed an increase of HO-1 expression in animals treated with DTA-1 at 72 h of reperfusion with significant differences. Thus, our results suggest that PC61 (anti-CD25) mAb treatment is deleterious, while DTA-1 (anti-GITR) mAb treatment presents a protective role in the renal IRI, indicating that some regulatory populations of T cells might have a role in IRI. (C) 2009 Elsevier B.V. All rights reserved.

Pro-oxidative synergic bactericidal effect of NO: kinetics and inhibition by nitroxides

NO plays diverse roles in physiological and pathological processes, occasionally resulting in opposing effects, particularly in cells subjected to oxidative stress. NO mostly protects eukaryotes against oxidative injury, but was demonstrated to kill prokaryotes synergistically with H2O2. This could be a promising therapeutic avenue. However, recent conflicting findings were reported describing dramatic protective activity of NO. The previous studies of NO effects on prokaryotes applied a transient oxidative stress while arbitrarily checking the residual bacterial viability after 30 or 60min and ignoring the process kinetics. If NO-induced synergy and the oxidative stress are time-dependent, the elucidation of the cell killing kinetics is essential, particularly for survival curves exhibiting a "shoulder" sometimes reflecting sublethal damage as in the linear-quadratic survival models. We studied the kinetics of NO synergic effects on H2O2-induced killing of microbial pathogens. A synergic pro-oxidative activity toward gram-negative and gram-positive cells is demonstrated even at sub-μM/min flux of NO. For certain strains, the synergic effect progressively increased with the duration of cell exposure, and the linear-quadratic survival model best fit the observed survival data. In contrast to the failure of SOD to affect the bactericidal process, nitroxide SOD mimics abrogated the pro-oxidative synergy of NO/H2O2. These cell-permeative antioxidants, which hardly react with diamagnetic species and react neither with NO nor with H2O2, can detoxify redox-active transition metals and catalytically remove intracellular superoxide and nitrogen-derived reactive species such as (•)NO2 or peroxynitrite. The possible mechanism underlying the bactericidal NO synergy under oxidative stress and the potential therapeutic gain are discussed.