leupeptins and Reperfusion-Injury

E3 ubiquitin ligase gene, WWP2, is associated with acute kidney injury (AKI). This research was conducted to explore the role of WWP2 in AKI. AKI cell model was produced in human renal proximal tubular epithelial cell line (HK-2) by ischemia-reperfusion (IR) injury. CCK8 and flow cytometry assay were performed to explore the influence of WWP2 overexpression on cell proliferation and apoptosis of IR-induced HK-2 cells. Quantitative real-time PCR and immunoblotting (IB) were performed to assess the gene and protein expression. Then, the influence of WWP2 on p53 ubiquitylation and degradation was estimated by immunoprecipitation assay. Our data indicated that WWP2 was down-regulated and p53 was up-regulated in IR-induced HK-2 cells. WWP2 overexpression promoted proliferation and inhibited apoptosis of IR-induced HK-2 cells. And WWP2 interacted with p53 and regulated p53 ubiquitylation and degradation. Furthermore, the influence of WWP2 on cell proliferation and apoptosis was rescued by MG132 (proteasome inhibitor) treatment. In conclusion, our work described for the first time the role of WWP2 in AKI, showing that WWP2 ameliorated AKI by mediating p53 ubiquitylation and degradation. Moreover, the study offers some important insights into the occurrence of AKI and WWP2 may be a novel target of AKI treatment. SIGNIFICANCE OF THE STUDY: Our data elaborates that WWP2 has protective effect against AKI by mediating p53 ubiquitylation and degradation. Thus, WWP2 might be a therapeutic target for AKI.

Topics: Acute Kidney Injury; Apoptosis; Cell Line; Cell Proliferation; Cell Survival; Flow Cytometry; HEK293 Cells; Humans; Kidney Tubules; Leupeptins; Real-Time Polymerase Chain Reaction; Reperfusion Injury; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases; Ubiquitination

Ischemia-reperfusion (I/R)-induced lung injury undermines lung transplantation (LTx) outcomes by predisposing lung grafts to primary graft dysfunction (PGD). Necrosis is a feature of I/R lung injury. However, regulated necrosis (RN) with specific signaling pathways has not been explored in an LTx setting. In this study, we investigated the role of RN in I/R-induced lung injury. To study I/R-induced cell death, we simulated an LTx procedure using our cell culture model with human lung epithelial (BEAS-2B) cells. After 18 h of cold ischemic time (CIT) followed by reperfusion, caspase-independent cell death, mitochondrial reactive oxygen species production, and mitochondrial membrane permeability were significantly increased. N-acetyl-Leu-Leu-norleucinal (ALLN) (calpain inhibitor) or necrostatin-1 (Nec-1) [receptor interacting serine/threonine kinase 1 (RIPK1) inhibitor] reduced these changes. ALLN altered RIPK1/RIPK3 expression and mixed lineage kinase domain-like (MLKL) phosphorylation, whereas Nec-1 did not change calpain/calpastatin expression. Furthermore, signal transducer and activator of transcription 3 (STAT3) was demonstrated to be downstream of calpain and regulate RIPK3 expression and MLKL phosphorylation during I/R. This calpain-STAT3-RIPK axis induces endoplasmic reticulum stress and mitochondrial calcium dysregulation. LTx patients' samples demonstrate that RIPK1, MLKL, and STAT3 mRNA expression increased from CIT to reperfusion. Moreover, the expressions of the key proteins are higher in PGD samples than in non-PGD samples. Cell death associated with prolonged lung preservation is mediated by the calpain-STAT3-RIPK axis. Inhibition of RIPK and/or calpain pathways could be an effective therapy in LTx.

Topics: Apoptosis; Calpain; Cells, Cultured; Humans; Imidazoles; Indoles; Leupeptins; Lung Transplantation; Necrosis; Phosphorylation; Primary Graft Dysfunction; Receptor-Interacting Protein Serine-Threonine Kinases; Receptors, Death Domain; Reperfusion Injury; Signal Transduction; STAT3 Transcription Factor

The activation of autophagy has been demonstrated to exert protective roles during hypoxia-reoxygenation (H/R)-induced brain injuries. This study aimed to investigate whether and how preconditioning with a proteasome inhibitor (MG-132), a proteasome promoter (Adriamycin, ADM), an autophagy inhibitor (3-methyladenine, 3-MA) and an autophagy promoter (Rapamycin, Rap) affected endoplasmic reticulum stress (ERS), the ubiquitin-proteasome system (UPS), autophagy, inflammation and apoptosis. Ubiquitin protein and 26S proteasome activity levels were decreased by MG-132 pretreatment but increased by ADM pretreatment at 2h, 4h and 6h following H/R treatment. MG-132 pretreatment led to the increased expression of autophagy-related genes, ER stress-associated genes and IκB but decreased the expression levels of NF-κB and caspase-3. ADM pretreatment led to the decreased expression of autophagy-related genes, ERS-associated genes and IκB but increased the expression of NF-κB and caspase-3. Pretreatment with 3-MA reduced the expression of autophagy-related genes, autophagy and UPS co-related genes, as well as apoptosis-related although the latter was increased by Rap pretreatment at 2h, 4h and 6h following H/R treatment. In vivo, pretreatment of rats with ADM, MG-132, 3-MA or Rap followed by ischemia-reperfusion (I/R) treatment resulted in similar changes. Proteasome inhibition preconditioning strengthened autophagy and ER stress but decreased apoptosis and inflammation. Autophagy promotion preconditioning exhibited similar changes. The combination of a proteasome inhibitor and an autophagy promoter might represent a new possible therapy to treat H/R or I/R injury-related diseases.

Topics: Adenine; Animals; Apoptosis; Autophagy; Cell Hypoxia; Cell Line; Cell Survival; Doxorubicin; Endoplasmic Reticulum Stress; Histone Deacetylase 6; Histone Deacetylases; Leupeptins; Lung; Male; NF-kappa B; Oxygen; Proteasome Endopeptidase Complex; Rats; Reperfusion Injury; Sirolimus; Ubiquitin

The dramatic shortage of organs leads to consider the steatotic livers for transplantation although their poor tolerance against ischemia reperfusion injury (IRI). Ubiquitin proteasome system (UPS) inhibition during hypothermia prolongs myocardial graft preservation. The role of UPS in the liver IRI is not fully understood. Bortezomib (BRZ) treatment at non-toxic doses of rats fed alcohol chronically has shown protective effects by increasing liver antioxidant enzymes. We evaluated and compared both proteasome inhibitors BRZ and MG132 in addition to University of Wisconsin preservation solution (UW) at low and non-toxic dose for fatty liver graft protection against cold IRI.. Steatotic and non-steatotic livers have been stored in UW enriched with BRZ (100 nM) or MG132 (25 μM), for 24h at 4°C and then subjected to 2-h normothermic reperfusion (37 °C). Liver injury (AST/ALT), hepatic function (bile output; vascular resistance), mitochondrial damage (GLDH), oxidative stress (MDA), nitric oxide (NO) (e-NOS activity; nitrates/nitrites), proteasome chymotrypsin-like activity (ChT), and UPS (19S and 20S5 beta) protein levels have been measured.. ChT was inhibited when BRZ and MG132 were added to UW. Both inhibitors prevented liver injury (AST/ALT), when compared to UW alone. BRZ increased bile production more efficiently than MG132. Only BRZ decreased vascular resistance in fatty livers, which correlated with an increase in NO generation (through e-NOS activation) and AMPK phosphorylation. GLDH and MDA were also prevented by BRZ. In addition, BRZ inhibited adiponectin, IL-1, and TNF alpha, only in steatotic livers.. MG132 and BRZ, administrated at low and non toxic doses, are very efficient to protect fatty liver grafts against cold IRI. The benefits of BRZ are more effective than those of MG132. This evidenced for the first time the potential use of UPS inhibitors for the preservation of marginal liver grafts and for future applications in the prevention of IRI.

Topics: Adiponectin; Animals; Boronic Acids; Bortezomib; Cold Ischemia; Cysteine Proteinase Inhibitors; Cytoprotection; Fatty Liver; Interleukin-1; Leupeptins; Liver Transplantation; Mitochondria, Liver; Organ Preservation; Organ Preservation Solutions; Oxidative Stress; Proteasome Inhibitors; Pyrazines; Rats; Rats, Zucker; Reperfusion Injury; Tumor Necrosis Factor-alpha

MG132 is a potent antioxidant and has been reported to play a protective role in ischemia/reperfusion (I/R) of many organs. Recent studies have shown that the Aryl hydrocarbon receptor (AhR) may play a beneficial role in I/R of many organs and an AhR agonist has been implicated in an anti-inflammatory role. MG132 might function as an AhR agonist through proteasome inhibition, possibly through the inhibition of NFκB. Herein, we hypothesized that MG132 may play a protective role in liver injury induced by intestinal I/R and we analyzed the expression behavior of AhR and NFκB to determine whether the two factors play a role in intestinal I/R.. Thirty-two Sprague-Dawley rats were divided into four groups: control, I/R, MG132 control, and MG132 pretreatment. The I/R and MG132 pretreatment groups were subjected to mesenteric arterial ischemia for 1 h and reperfusion for 3 h. The control and MG132 control groups underwent surgical preparation including isolation of the superior mesenteric artery (SMA) without occlusion. The MG132 control and MG132 pretreatment groups were subjected to intraperitoneal administration of 0.5 mg/kg MG132 30 min before surgery. We collected serum specimens to measure TNF-α, IL-6, liver tissue levels of malondialdehyde (MDA), AhR, and cyp1a2; NFκB, IκBα, and ICAM-1 were also tested. Histologic changes of liver and intestine were subsequently evaluated.. Compared with the control group, significant increases in MDA, NFκB, and ICAM-1 levels were accompanied by decreases in AhR, cyp1a2, and IκBα expression in the liver in the I/R group, which is consistent with liver and intestinal tissue injury. MG132 blocked the alterations of the indicators above. There were no changes in the MG132 control group compared with the control group in the indicators above.. This study demonstrated that MG132 has a significant effect in protection against liver injury induced by intestinal I/R, which may be due to modulation of the AhR and NFκB pathways.

Topics: Animals; Antioxidants; Cytochrome P-450 CYP1A2; Intercellular Adhesion Molecule-1; Interleukin-6; Leupeptins; Liver; Male; Malondialdehyde; Models, Animal; NF-kappa B; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Aryl Hydrocarbon; Reperfusion Injury; Signal Transduction; Tumor Necrosis Factor-alpha

Acute kidney dysfunction after ischemia-reperfusion injury (IRI) may be a consequence of persistent intrarenal vasoconstriction. Regulators of G-protein signaling (RGSs) are GTPase activators of heterotrimeric G proteins that can regulate vascular tone. RGS4 is expressed in vascular smooth muscle cells in the kidney; however, its protein levels are low in many tissues due to N-end rule-mediated polyubiquitination and proteasomal degradation. Here, we define the role of RGS4 using a mouse model of IRI comparing wild-type (WT) with RGS4-knockout mice. These knockout mice were highly sensitized to the development of renal dysfunction following injury exhibiting reduced renal blood flow as measured by laser-Doppler flowmetry. The kidneys from knockout mice had increased renal vasoconstriction in response to endothelin-1 infusion ex vivo. The intrinsic renal activity of RGS4 was measured following syngeneic kidney transplantation, a model of cold renal IRI. The kidneys transplanted between knockout and WT mice had significantly reduced reperfusion blood flow and increased renal cell death. WT mice administered MG-132 (a proteasomal inhibitor of the N-end rule pathway) resulted in increased renal RGS4 protein and in an inhibition of renal dysfunction after IRI in WT but not in knockout mice. Thus, RGS4 antagonizes the development of renal dysfunction in response to IRI.

Topics: Acute Kidney Injury; Animals; Cysteine Proteinase Inhibitors; Disease Models, Animal; Endothelin-1; Enzyme Activation; GTP-Binding Protein alpha Subunits, Gq-G11; Kidney; Kidney Transplantation; Laser-Doppler Flowmetry; Leupeptins; Ligation; Male; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Nephrectomy; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Renal Circulation; Reperfusion Injury; RGS Proteins; Time Factors; Vasoconstriction; Vasoconstrictor Agents

We previously demonstrated that endogenous interleukin-6 (IL-6) is upregulated and may be neuroprotective after retinal ischemia. The purpose of this study is to investigate the role of nuclear factor kappa-B (NF-kappaB) in regulating IL-6 expression after ischemia. NF-kappaB p65 mRNA levels were significantly elevated between 2 and 12 h after the insult. A high number of NF-kappaB p65 positive cells were detected in the inner retina at 12 h after ischemia. Activated nuclear NF-kappaB p65 and IL-6 were colocalized in cells, which were also marked by a microglial/phagocytic cell marker (ED1) in the inner retina. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132, a proteasome inhibitor, which inhibits IkappaB degradation and hence prevents the activation and translocation of NF-kappaB into the nucleus) abolished the increase in NF-kappaB p65 mRNA levels after the insult, while there was no effect by helenalin (an inhibitor which inhibits NF-kappaB activity by alkylation of the p65 subunit, thereby blocking its binding to the target DNA). However, MG-132 and/or helenalin significantly diminished the increase in IL-6 mRNA levels after the insult. Small interfering RNAs (siRNAs, inhibit target gene expression through the sequence-specific destruction of the target messenger RNA) against NF-kappaB p65 significantly reduced the increase in NF-kappaB p65 mRNA levels as well as IL-6 mRNA levels after ischemia. The number of retinal ganglion cells (RGCs) was also significantly decreased using the inhibitors of NF-kappaB compared with those of the controls after ischemia. These findings support the hypothesis that upregulation of endogenous retinal IL-6 in retinal I/R injury in microglial/phagocytic cells is controlled predominantly by NF-kappaB p65.

Topics: Analysis of Variance; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Count; Cysteine Proteinase Inhibitors; Drug Interactions; Gene Expression Regulation; Immunohistochemistry; Interleukin-6; Leupeptins; Male; NF-kappa B; Rats; Rats, Inbred Lew; Reperfusion Injury; Retina; Retinal Ganglion Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Sesquiterpenes; Sesquiterpenes, Guaiane; Time Factors

Although ischemia reperfusion (I/R) induces apoptotic damage of mammalian small intestine, the molecular mechanism is largely unknown. We investigated the appearance of apoptosis at various time-points (0-24 h) of reperfusion after 1-h ischemia and the expression of various apoptosis-related proteins, such as Bcl-2, Bax, Fas, Fas ligand (FasL), activated caspase-3, and cytochrome c, immunohistochemically in rat small intestine. As assessed by TUNEL and electron microscopy, apoptotic cells were increased at 3 h of reperfusion in all intestinal parts (villous epithelium, crypt epithelium, and stroma of intestine). Moreover, the TUNEL-positive cells in the stroma were later identified as T cells. The expression of Fas and FasL as well as activated caspase-3 was markedly increased at 3 h of reperfusion in the stroma. In the villous epithelium, a transient decrease in Bcl-2 expression was found while in the crypt epithelium, Fas expression was induced. Finally, intraperitoneal injection of leupeptin (an SH-protease inhibitor) after I/R resulted in a significant inhibition of the induction of apoptosis in the stroma and crypt epithelium. Our results indicate that the triggering molecules of apoptosis in the I/R rat small intestine may vary depending on cell type and that the use of a broad-spectrum protease inhibitor may reduce intestinal damage.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase Inhibitors; Caspases; Cytochromes c; Fas Ligand Protein; Immunohistochemistry; In Situ Nick-End Labeling; Intestine, Small; Leupeptins; Membrane Glycoproteins; Microscopy, Electron; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reperfusion Injury

Topics: Animals; Female; Leupeptins; Liver; NF-kappa B; Rats; Rats, Wistar; Reperfusion Injury

Ca(2+) overload and free-radical injury are two mutually non-exclusive phenomena suggested to cause myocardial ischemia-reperfusion (IR)-induced contractile dysfunction; however, the mechanisms underlying their effects are not clear. One possible mechanism is the proteolytic modification of proteins by Ca(2+)-dependent proteases, such as calpains, which are activated during Ca(2+) overload that occurs in IR. The sarcoplasmic reticulum (SR) plays a central role in mediating cardiac contractility and therefore any impairment in SR function will induce cardiac contractile dysfunction. We therefore investigated the possibility whether SR proteins were the target for calpain action in IR. Langendorff-perfused rat hearts were subjected to IR in the presence and absence of leupeptin, a calpain inhibitor and the effects of calpain inhibition was examined on cardiac performance, SR function, and its regulation by protein phosphorylation as well as expression of SR Ca(2+)-cycling and -regulatory proteins. Our results show a depression in cardiac contractile function and activation of calpain during IR. Treatment with leupeptin recovered cardiac contractile function and attenuated calpain activity in IR hearts. The cardioprotection observed upon leupeptin treatment was associated with improved SR function and regulation. The recovery in SR function and regulation was consistent with prevention of IR-induced decrease in the expression of key SR Ca(2+)-handling and -regulatory proteins. Our results suggest that a downregulation of SR proteins by calpain may be a mechanism by which Ca(2+) overload causes cardiac contractile dysfunction during IR.

Topics: Animals; Blotting, Western; Calcium; Calcium-Binding Proteins; Calcium-Transporting ATPases; Calpain; Calsequestrin; Cyclic AMP-Dependent Protein Kinases; Cytosol; Down-Regulation; Leupeptins; Male; Myocardial Contraction; Myocardium; Perfusion; Phosphorylation; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Time Factors

The objectives of this study were to 1) determine the time-course of T-lymphocyte adhesion to monolayers of human umbilical vein endothelial cell (HUVEC) that were exposed to 60 min of anoxia followed by 24 h of reoxygenation, and 2) define the mechanisms responsible for the hyperadhesivity of postanoxic HUVEC to human T-lymphocytes.. Human peripheral blood mononuclear leukocytes were isolated from heparinized peripheral blood. T-lymphocytes were obtained by negative selection using a MACS column. HUVEC monolayers were exposed to anoxia/reoxygenation (A/R), and then reacted with 51Cr -labeled T-lymphocytes in adhesion assays.. A/R leads to an increased adhesion of T-lymphocytes to HUVEC monolayers, with peak responses occurring at 8 h after reoxygenation. This adhesion response was largely attributed to the CD4+ T-cell subset. The hyperadhesivity of A/R-exposed HUVEC was inhibited by monoclonal antibodies directed against either LFA-1, VLA-4, ICAM-1, or VCAM-1, indicating a contribution of these adhesion molecules and their ligands. Moreover, T-cell hyperadhesivity was attenuated by anti- IL-8. consistent with a role for this chemokine in the adhesion response. Protein synthesis inhibitors (actinomycin D and cycloheximide) as well as chemical inhibitors of (and binding ds-oligonucleotides to) NFkappaB and AP-1 significantly attenuated the A/R-induced T-lymphocyte adhesion responses. The kinetics of VCAM-1 on post-anoxic HUVEC correlated with the T-lymphocyte adhesion response.. A/R elicits a T-lymphocyte-endothelial cell adhesion response that involves transcription-dependent surface expression of VCAM-1.

Topics: Antibodies, Monoclonal; Benzamides; Cell Adhesion; Cell Adhesion Molecules; Cell Hypoxia; Cells, Cultured; Cysteine Endopeptidases; Endothelium, Vascular; Humans; Interleukin-8; Ischemia; Kinetics; Leupeptins; Multienzyme Complexes; NF-kappa B; Oxygen; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Reperfusion Injury; T-Lymphocyte Subsets; Thionucleotides; Time Factors; Transcription Factor AP-1; Tumor Necrosis Factor-alpha; Umbilical Veins; Vascular Cell Adhesion Molecule-1

Protease inhibitors attenuate ischemia/reperfusion injury. However, the underlying mechanisms by which protease inhibitors prevent reperfusion injury remain obscure. Neutrophils play an important role in reperfusion injury. We studied the effects of urinary trypsin inhibitor (UTI) on production of the C-X-C chemokine, cytokine-induced neutrophil chemoattractant (CINC), by Kupffer cells during ischemia/reperfusion of the liver.. Liver ischemia was induced in rats by occlusion of the portal vein for 30 min. UTI (50,000 U/kg) was injected intravenously 5 min before vascular clamping. Serum CINC concentrations were measured by enzyme-linked immunosorbent assay. Levels of CINC mRNA in the liver were determined by Northern blot analysis. We also examined the inhibitory effects of UTI on in vitro CINC production by peritoneal macrophages in response to neutrophil elastase (NE).. Serum CINC concentrations increased and peaked 6 h after reperfusion. However, pretreatment of animals with UTI blunted this increase in CINC and significantly reduced CINC mRNA levels in the liver after ischemia/reperfusion. UTI also decreased neutrophil accumulation in the liver 24 h after reperfusion. In vitro CINC production by Kupffer cells from rats pretreated with UTI 3 h after ischemia/reperfusion was significantly decreased compared to those from untreated animals. UTI reduced NE activity in vitro in a dose-dependent manner, and UTI significantly reduced in vitro CINC production by peritoneal macrophages stimulated with NE.. UTI reduces the production of CINC by Kupffer cells stimulated with NE, attenuating ischemia/reperfusion injury of the liver.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cells, Cultured; Chemokine CXCL1; Chemokines, CXC; Chemotactic Factors; Gene Expression Regulation; Glycoproteins; Growth Substances; Humans; Injections, Intravenous; Intercellular Signaling Peptides and Proteins; Kupffer Cells; Leukocyte Elastase; Leupeptins; Liver; Macrophages, Peritoneal; Male; Portal Vein; Protease Inhibitors; Rats; Rats, Wistar; Reperfusion Injury; RNA, Messenger; Transcription, Genetic; Trypsin Inhibitors; Tumor Necrosis Factor-alpha