Alcohol dehydrogenase 1 is a tubular mitophagy-dependent apoptosis inhibitor against septic acute kidney injury.

Alcohol dehydrogenase 1 (ADH1) is an alcohol-oxidizing enzyme with poorlydefined biology. Here we report that ADH1 is highly expressed in kidneys of mice with lethal endotoxemia and is transcriptionally upregulated in tubular cells by lipopolysaccharide (LPS) stimuli through TLR4/NF-κB cascade. The Adh1 knockout (Adh1KO) mice with lethal endotoxemia displayed increased susceptibility to acute kidney injury (AKI) but not systemic inflammatory response. Adh1KO mice develop more severe tubular cell apoptosis in comparison to Adh1 wild-type (Adh1WT) mice during course of lethal endotoxemia. ADH1 deficiency facilitates the LPS-induced tubular cell apoptosis in a caspase-dependent manner. Mechanistically, ADH1 deficiency dampens tubular mitophagy that relies on PINK1-Parkin pathway characterized by the reduced membrane potential, reactive oxygen species (ROS) and release of fragmented mtDNA to cytosol. Kidney-specific overexpression of PINK1 and Parkin by adeno-associated viral vector 9 (AAV9) delivery ameliorates AKI exacerbation in Adh1KO mice with lethal endotoxemia. Our study supports the notion that ADH1 is critical for blockade of tubular apoptosis mediated by mitophagy, allowing the rapid identification and targeting of alcohol-metabolic route applicable to septic AKI.

Auto- and paracrine rewiring of NIX-mediated mitophagy by insulin-like growth factor-binding protein 7 in septic AKI escalates inflammation-coupling tubular damage.

AIMS: Inflammation-coupling tubular damage (ICTD) contributes to pathogenesis of septic acute kidney injury (AKI), in which insulin-like growth factor-binding protein 7 (IGFBP-7) serves as a biomarker for risk stratification. The current study aims to discern how IGFBP-7 signalling influences ICTD, the mechanisms that underlie this process and whether blockade of the IGFBP-7-dependent ICTD might have therapeutic value for septic AKI. MATERIALS AND METHODS: In vivo characterization was carried out in B6/JGpt-Igfbp7em1Cd1165/Gpt mice subjected to cecal ligation and puncture (CLP). Transmission electron microscopy, immunofluorescence, flow cytometry, immunoblotting, ELISA, RT-qPCR and dual-luciferase reporter assays were used to determine mitochondrial functions, cell apoptosis, cytokine secretion and gene transcription. KEY FINDINGS: ICTD augments the transcriptional activity and protein secretion of tubular IGFBP-7, which enables an auto- and paracrine signalling via deactivation of IGF-1 receptor (IGF-1R). Genetic knockout (KO) of IGFBP-7 provides renal protection, improves survival and resolves inflammation in murine models of cecal ligation and puncture (CLP), while administering recombinant IGFBP-7 aggravates ICTD and inflammatory invasion. IGFBP-7 perpetuates ICTD in a NIX/BNIP3-indispensable fashion through dampening mitophagy that restricts redox robustness and preserves mitochondrial clearance programs. Adeno-associated viral vector 9 (AAV9)-NIX short hairpin RNA (shRNA) delivery ameliorates the anti-septic AKI phenotypes of IGFBP-7 KO. Activation of BNIP3-mediated mitophagy by mitochonic acid-5 (MA-5) effectively attenuates the IGFBP-7-dependent ICTD and septic AKI in CLP mice. SIGNIFICANCE: Our findings identify IGFBP-7 is an auto- and paracrine manipulator of NIX-mediated mitophagy for ICTD escalation and propose that targeting the IGFBP-7-dependent ICTD represents a novel therapeutic strategy against septic AKI.

Interruption of neutrophil extracellular traps formation dictates host defense and tubular HOXA5 stability to augment efficacy of anti-Fn14 therapy against septic AKI.

The immunosuppressive, inflammatory microenvironment orchestrated by neutrophil extracellular traps (NETs) plays a principal role in pathogenesis of sepsis. Fibroblast growth factor-inducible molecule 14 (Fn14) has been established as a potential target for septic acute kidney injury (AKI), making further therapeutic benefits from combined NETs and Fn14 blockade possible. Methods: The concurrence of NETs and Fn14 in mice and patients with septic AKI were assessed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA) and in silico studies. Survival, histopathological and biochemical analyses of wild-type and PAD4-deficient CMV-Cre; PAD4 fl/fl mice with septic AKI were applied to evaluate the efficacy of either pharmacological or genetic NETs interruption in combination with Fn14 blockade. Molecular mechanisms underlying such effects were determined by CRISPR technology, fluorescence-activated cell sorter analysis (FACS), cycloheximide (CHX) pulse-chase, luciferase reporter and chromatin immunoprecipitation (ChIP) assay. Results: NETs formation is concurred with Fn14 upregulation in murine AKI models of abdominal, endotoxemic, multidrug-resistant sepsis as well as in serum samples of patients with septic AKI. Pharmacological or genetic interruption of NETs formation synergizes with ITEM-2, a monoclonal antibody (mAb) of Fn14, to prolong mice survival and provide renal protection against abdominal sepsis, the effects that could be abrogated by elimination of macrophages. Interrupting NETs formation predominantly perpetuates infiltration and survival of efferocytic growth arrest-specific protein 6+ (GAS6+) macrophages in combination with ITEM-2 therapy and enhances transcription of tubular cell-intrinsic Fn14 in a DNA methyltransferase 3a (DNMT3a)-independent manner through dismantling the proteasomes-mediated turnover of homeobox protein Hox-A5 (HOXA5) upon abdominal sepsis challenge or LPS stimuli. Pharmacological NETs interruption potentiates the anti-septic AKI efficacy of ITEM-2 in murine models of endotoxemic and multidrug-resistant sepsis. Conclusion: Our preclinical data propose that interrupting NETs formation in combination with Fn14 mAb might be a feasible therapeutic strategy for septic AKI.

Redox DAPK1 destabilizes Pellino1 to govern inflammation-coupling tubular damage during septic AKI.

Tubular damage initiated by inflammatory response and ischemic/hypoxic stress is a hallmark of septic acute kidney injury (AKI), albeit the molecular mechanism coupling the two events remains unclear. We investigated the intrinsic nature of tubular damage with respect to inflammatory/hypoxic stress during septic AKI. Methods: The apoptotic response of tubular cells to LPS stimuli was analyzed before and after hypoxia exposure. Cellular ubiquitination, co-immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and CRISPR technology were adopted to determine the molecular mechanism underlying this process. In vivo characterization was performed in wild-type and DAPK1-/- mice models of cecal ligation and puncture (CLP). Results: We found that the MyD88-dependent inflammatory response couples to tubular damage during LPS stimuli under hypoxia in a Fn14/SCFFbxw7α-dispensable manner via recruitment of caspase-8 with TRIF-RIP1 signalosome mediated by DAPK1, which directly binds to and phosphorylates Pellino1 at Ser39, leading to Pellino1 poly-ubiquitination and turnover. Either pharmacological deactivation or genetic ablation of DAPK1 makes tubular cells refractory to the LPS-induced damage in the context of hypoxia, while kinase activity of DAPK1 is essential for ruin execution. Targeting DAPK1 effectively protects mice against septic AKI and potentiates the efficacy of a MyD88 homodimerization inhibitor, ST2825. Conclusion: Our findings provide a rationale for the mechanism whereby inflammation intersects with hypoxic tubular damage during septic AKI through a previously unappreciated role of DAPK1-inducible Ser39 phosphorylation in Pellino1 turnover and underscore that combined targeting DAPK1 and MyD88 might be a feasible strategy for septic AKI management.

MicroRNA-19a Targets Fibroblast Growth Factor-Inducible Molecule 14 and Prevents Tubular Damage in Septic AKI.

Fibroblast growth factor-inducible molecule 14 (Fn14) plays a principal role in triggering tubular damage during septic acute kidney injury (AKI). Here, we explore the mechanism underlying Fn14 deregulation in septic AKI. We identify Fn14 as a bona fide target of miR-19a, which directly binds to 3' UTR of Fn14 for repression independent of cylindromatosis (CYLD), the deubiquitinase (DUB) downstream of miR-19a, and thereby antagonizes the LPS-induced tubular cell apoptosis. Genetic ablation of Fn14, but not of CYLD, abolishes the ability of miR-19a to antagonize the tubular apoptosis by lipopolysaccharide (LPS). In mice, systemic delivery of miR-19a confers protection against septic AKI. Our findings implicate that miR-19a may serve as a promising therapeutic candidate in the prevention of septic AKI.

Regulation of Fn14 stability by SCFFbxw7α during septic acute kidney injury.

Acute kidney injury (AKI) initiated by sepsis remains a thorny problem despite recent advancements in its clinical management. Having been found to be activated during AKI, fibroblast growth factor-inducible molecule 14 (Fn14) may be a potential therapeutic target because of its involvement in the molecular basis of injury. Here, we report that LPS induces apoptosis of mouse cortical tubule cells mediated by Fn14, for which simultaneous Toll-like receptor (TLR)4 activation is required. Mechanistically, TLR4 activation by lipopolysaccharide, through disassociating E3 ligase SCFFbxw7α from Fn14, dismantles Lys48-linked polyubiquitination of Fn14 and stabilizes it. Pharmacological deactivation of Fn14 with monoclonal antibody ITEM-2 provides effective protection against lethal sepsis and AKI in mice. Our study underscores an adaptive mechanism whereby TLR4 regulates SCFFbxw7α-dependent Fn14 stabilization during inflammatory tubular damage and further supports investigation of targeting Fn14 in clinical trials of patients with septic AKI.

Productive transcription of miR-124-3p by RelA and RNA polymerase II directs RIP1 ubiquitination-dependent apoptosis resistance during hypoxia.

The K63-linked ubiquitination of RIP1 coordinates survival/death homeostasis by driving transcription of genes downstream of RelA. Previously, we demonstrated that EGF-dependent RelA transactivation overcomes hypoxia-initiated apoptosis, yet the underlying mechanisms remain mysterious. We report here that UBXN1 deficiency empowers apoptosis resistance against hypoxia through triggering IκBα degradation, for which K63-linked ubiquitination of RIP1 is required. MiR-124-3p is a bona fide inhibitor upstream of UBXN1, thereby antagonizing the hypoxia-initiated apoptosis. UBXN1 repression by miR-124-3p restores the K63-linked ubiquitination of RIP1, IKKß phosphorylation, IκBα-RelA disassembly, RelA nuclear localization and transactivation of EGF gene as well as EGF secretion under hypoxia. Reconstitution of wild-type UBXN1, but not a truncated UBXN1ΔUBA mutant, or pharmacological inhibition of RelA transactivation in miR-124-3p-replete cells compromises the apoptosis-resistant phenotypes of miR-124-3p. Hypoxia transcriptionally downregulates miR-124-3p by disassociating RelA and RNAP II from its promoter. EGFR activation renders the K63-linked ubiquitination of RIP1 and hypoxic tolerance in conjunction with miR-124-3p. Our findings identify a pivotal role of miR-124-3p in ubiquitin conjugation of RIP1 against hypoxic damage and underscore that productive transcription of miR-124-3p by RelA and RNAP II might be a switching mechanism for this process.

Pharmacological Inhibition of PTEN Restores Remote Ischemic Postconditioning Cardioprotection in Hypercholesterolemic Mice: Potential Role of PTEN/AKT/GSK3ß SIGNALS.

Although remote ischemic postconditioning (RIPC) was shown to confer cardioprotection against myocardial ischemia/reperfusion (I/R) injury in normal animals, whether RIPC-induced cardioprotection is altered in the presence of hypercholesterolemia, a comorbidity with acute myocardial infarction (AMI) patients has yet to be determined. Normal or 2% cholesterol chow was fed to male C57BL/6J mice for 12 weeks to induce hypercholesterolemia, then normal or hypercholesterolemic murine hearts were exposed to AMI by coronary artery ligation. RIPC was induced by four episodes of 5 min femoral artery occlusion followed by 5 min reperfusion immediately after myocardial reperfusion in mice. Following I/R, RIPC significantly attenuated postischemic infarct size, hindered cardiomyocyte apoptosis, improved cardiac systolic function, decreased phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression, and further increased Akt and GSK-3ß phosphorylation in non-hypercholesterolemic, but not in hypercholesterolemic mice. Application of the PTEN inhibitor bisperoxovanadium (BpV) (1.0 mg/kg) reduced postischemic infarct size, attenuated cardiomyocyte apoptosis, and improved cardiac dysfunction in normal, but not in hypercholesterolemic mice. Further, increased dose of BpV (2 mg/kg or 10 mg/kg) failed to rescue the detrimental effects of hypercholesterolemia on I/R in mice following I/R. Especially important, we demonstrated that the combination BpV and RIPC exerted marked cardioprotective effects both in normal and hypercholesterolemic mice with I/R, indicating that PTEN inhibition restores RIPC-elicited myocardial protection in the presence of hypercholesterolemia. Our results demonstrated that hypercholesterolemia attenuated RIPC-induced cardioprotection against I/R injury by alteration of PTEN/Akt/GSK3ß signals, and inhibition of PTEN rescued RIPC-induced cardioprotection in the presence of hypercholesterolemia.

Transcriptional downregulation of microRNA-19a by ROS production and NF-κB deactivation governs resistance to oxidative stress-initiated apoptosis.

Cell apoptosis is one of the main pathological alterations during oxidative stress (OS) injury. Previously, we corroborated that nuclear factor-κB (NF-κB) transactivation confers apoptosis resistance against OS in mammalian cells, yet the underlying mechanisms remain enigmatic. Here we report that microRNA-19a (miR-19a) transcriptionally regulated by reactive oxygen species (ROS) production and NF-κB deactivation prevents OS-initiated cell apoptosis through cylindromatosis (CYLD) repression. CYLD contributes to OS-initiated cell apoptosis, for which NF-κB deactivation is essential. MiR-19a directly represses CYLD via targeting 3' UTR of CYLD, thereby antagonizing OS-initiated apoptosis. CYLD repression by miR-19a restores the IKKß phosphorylation, RelA disassociation from IκBα, IκBα polyubiquitination and degradation, RelA recruitment at VEGF gene promoter as well as VEGF secretion in the context of OS. Either pharmacological deactivation of NF-κB or genetic upregulation of CYLD compromises the apoptosis-resistant phenotypes of miR-19a. Furthermore, miR-19a is transcriptionally downregulated upon OS in two distinct processes that require ROS production and NF-κB deactivation. VEGF potentiates the ability of miR-19a to activate NF-κB and render apoptosis resistance. Our findings underscore a putative mechanism whereby CYLD repression-mediated and NF-κB transactivation-dependent miR-19a regulatory feedback loop prevents cell apoptosis in response to OS microenvironment.

MicroRNA-130b transcriptionally regulated by histone H3 deacetylation renders Akt ubiquitination and apoptosis resistance to 6-OHDA.

Apoptosis of DA neurons is a contributing cause of disability and death for Parkinson's disease (PD). Akt may become a potential therapeutic target for PD since Akt has been deactivated during DA neuron apoptosis. We previously demonstrated that Akt confers apoptosis resistance against 6-OHDA in DA neuron-like PC12 cells, yet the underlying mechanisms accounted for this are not fully understood. Here we report that microRNA-130b (miR-130b)-dependent and cylindromatosis (CYLD) repression-mediated Akt ubiquitination renders apoptosis resistance of PC12 cells to 6-OHDA, which elicits histone H3 deacetylation-induced transcriptional downregulation of miR-130b vice versa. CYLD deficiency ubiquitinates Akt at Lys63, thereby phosphorylating Akt and antagonizing 6-OHDA-initiated apoptosis. MiR-130b targetedly represses CYLD and increases apoptosis resistance to 6-OHDA. CYLD repression by miR-130b restores Akt ubiquitination and activation, GSK3ß and FoxO3a phosphorylation, FoxO3a removal from Bim promoter as well as Bim downregulation during 6-OHDA administration. CYLD deficiency-mediated Akt activation is instrumental for the apoptosis-resistant phenotypes of miR-130b. In addition, 6-OHDA transcriptionally downregulates miR-130b through recruitment of HDAC3 at the promoter. Furthermore, EPO potentiates the ability of miR-130b to activate Akt and augment apoptosis resistance. Our findings identify the apoptosis-resistant function of miR-130b and suggest that histone H3 deacetylation plays a pivotal role in regulating miR-130b transcription in response to 6-OHDA.

Hypercholesterolemia Abrogates Remote Ischemic Preconditioning-Induced Cardioprotection: Role of Reperfusion Injury Salvage Kinase Signals.

Remote ischemic preconditioning (RIPC) is one of the most powerful intrinsic cardioprotective strategies discovered so far and experimental data indicate that comorbidity may interfere with the protection by RIPC. Therefore, we investigate whether RIPC-induced cardioprotection was intact in hypercholesterolemic rat hearts exposed to ischemia reperfusion in vivo. Normal or hypercholesterolemic rat hearts were exposed to 30 min of ischemia and 2 h of reperfusion, with or without RIPC, PI3K inhibitor wortmannin, MEK-ERK1/2 inhibitor PD98059, GSK3ß inhibitor SB216763. Infarct size, apoptosis, MG53, PI3K-p85, p-Akt, p-ERK1/2, p-GSK3ß, and cleaved Caspase-3 were determined. RIPC reduced infarct size, limited cardiomyocyte apoptosis following IR that was blocked by wortmannin but not PD98059. RIPC triggered unique cardioprotective signaling including MG53, phosphorylation of Akt, and glycogen synthase kinase-3ß (GSK3ß) in concert with reduced proapoptotic active caspase-3. In contrast, RIPC failed to reduce myocardial necrosis and apoptosis as well as to increase the phosphorylated Akt and GSK3ß in hypercholestorolemic myocardium. Importantly, we found that inhibition of GSK with SB216763 reduced myocardial infarct size in healthy and hypercholesterolemic hearts, but no additional cardioprotective effect was achieved when combined with RIPC. Our results suggest that acute GSK3ß inhibition may provide a novel therapeutic strategy for hypercholesterolemic patients during acute myocardial infarction, whereas RIPC is less effective due to signaling events that adversely affect GSK3ß.

NF-κB-dependent transcriptional upregulation of cyclin D1 exerts cytoprotection against hypoxic injury upon EGFR activation.

Apoptosis of neural cells is one of the main pathological features in hypoxic/ischemic brain injury. Nuclear factor-κB (NF-κB) might be a potential therapeutic target for hypoxic/ischemic brain injury since NF-κB has been found to be inactivated after hypoxia exposure, yet the underlying molecular mechanisms of NF-κB inactivation are largely unknown. Here we report that epidermal growth factor receptor (EGFR) activation prevents neuron-like PC12 cells apoptosis in response to hypoxia via restoring NF-κB-dependent transcriptional upregulation of cyclin D1. Functionally, EGFR activation by EGF stimulation mitigates hypoxia-induced PC12 cells apoptosis in both dose- and time-dependent manner. Of note, EGFR activation elevates IKKß phosphorylation, increases IκBα ubiquitination, promotes P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as upregulates cyclin D1 expression. EGFR activation also abrogates the decrease of IKKß phosphorylation, reduction of IκBα ubiquitination, blockade of P65 nuclear translocation and recruitment at cyclin D1 gene promoter as well as downregulation of cyclin D1 expression induced by hypoxia. Furthermore, NF-κB-dependent upregulation of cyclin D1 is instrumental for the EGFR-mediated cytoprotection against hypoxic apoptosis. In addition, the dephosphorylation of EGFR induced by either EGF siRNA transfection or anti-HB-EGF neutralization antibody treatment enhances hypoxic cytotoxicity, which are attenuated by EGF administration. Our results highlight the essential role of NF-κB-dependent transcriptional upregulation of cyclin D1 in EGFR-mediated cytoprotective effects under hypoxic preconditioning and support further investigation of EGF in clinical trials of patients with hypoxic/ischemic brain injury.

VEGF-mediated NF-κB activation protects PC12 cells from damage induced by hypoxia.

Neuronal apoptosis is a contributing cause of disability and death in cerebral ischemia. Nuclear factor-κB (NF-κB) may become a potential therapeutic target for hypoxic/ischemic neuron damage because NF-κB is inactivated after hypoxia exposure. Vascular endothelial growth factor (VEGF) has been found to improve neurological function recovery in cerebral ischemic injury although the exact molecular mechanisms that underlie the neuroprotective function of VEGF remain largely unknown. Here we defined the mechanism by which VEGF antagonized neuron-like PC12 cells apoptosis induced by hypoxia mimetic agent cobalt chloride (CoCl2) is through restoration of NF-κB activity. Depletion of VEGF with small interfering RNA (siRNA) in PC12 cells conferred CoCl2-induced cytotoxicity which was mitigated by VEGF administration. Treatment of PC12 cells with VEGF attenuated the CoCl2-induced cytotoxicity in both dose- and time-dependent manner. Mechanistically, VEGF increased IκBα phosphorylation and ubiquitination, promoted P65 nuclear translocation as well as upregulated XIAP and CCND1 expression. Meanwhile, VEGF administration reversed the dysregulation of IκBα phosphorylation and ubiquitination, P65 nuclear translocation as well as XIAP and CCND1 expression induced by CoCl2. Notably, the VEGF-dependent cytoprotection was abolished by pretreatment with BAY 11-7085, a specific inhibitor of NF-κB. Our data suggest that VEGF/NF-κB signalling pathway represents an adaptive mechanism that protects neural cells against hypoxic damage.

[cAMP response element binding protein expression and activity in acute lung injury induced by endotoxinemia following hemorrhagic shock: experiment with rabbits].

OBJECTIVE: To investigate the cAMP response element binding protein (CREB) expression and activity in acute lung injury induced by endotoxinemia following hemorrhagic shock. METHODS: Thirty-six rabbits were randomly divided into 3 equal groups: 2 hours after the endotoxinemia following hemorrhagic shock group that underwent exsanguination, injected intraperitoneally with lipopolysaccharide (LPS) after the blood pressure became stable, and then underwent extraction of carotid blood for blood gas test and killed 2 hours later; 12 hours after the endotoxinemia following hemorrhagic shock (two hits) group: undergoing the above mentioned management and being killed 12 hours later; and control group, undergoing sham operation. The lungs were taken out to undergo histological examination. The partial arterial blood pressure of oxygen (PaO2) and wet lung weight to dry lung weight ratio (W/D) were measured. The concentration of tumor necrosis factor (TNF)-alpha in the lung homogenate was measured by enzyme-linked immunosorbent assay (ELISA). The expression of CREB in the lung was assessed by Western blotting and the CREB/DNA binding activity was assayed with electrophoretic mobility shift assay (EMSA). RESULTS: Pathological examination of the lung tissues showed that the alveolar structures were severely destroyed and large number of WBC infiltrating in both alveolar sacs and pulmonary interstitial with RBC leakage the 12 hours after the endotoxinemia following hemorrhagic shock group, and the changes were mild in the 2 hours after two hits group. The PaO2 was significantly reduced in the 12 hour after endotoxinemia following hemorrhagic shock group compared with the control group (P < 0.01). The lung W/D and the concentration of TNF-alpha in lung homogenate were significantly increased in the 12 hours after endotoxinemia following hemorrhagic shock group compared with the control group (both P < 0.01). Nevertheless there were not significant differences in the PaO2, W/D and TNF-alpha between the 2 hours after two hits group and the control group. The expression level and DNA binding activity of CREB in the lung were significantly higher in both 2 and 12 hours after endotoxinemia following hemorrhagic shock groups than those in the control group (all P < 0.01). CONCLUSION: CREB in the lung is activated by endotoxinemia following hemorrhagic shock and may participate in the inflammatory response in acute lung injury by regulating the expression of inflammatory factors.