Many receptors involved with innate immunity activate the inhibitor kappa B kinase signalosome (IKK). The active complex appears to be assembled from the two kinase units, IKKα and IKKβ with the ...regulatory protein NEMO. Because we previously found that RNA silencing of clathrin heavy chains (CHC), in transformed human lung pneumocytes (A549), decreased TNFα‐induced signaling and phosphorylation of inhibitor kappa B (IκB), we hypothesized that CHC forms cytoplasmic complexes with members of the IKK signalosome. Widely available antibodies were used to immunoprecipitate IKKα and NEMO interactomes. Analysis of the affinity interactomes by mass spectrometry detected clathrin with both baits with high confidence. Using the same antibodies for indirect digital immunofluorescence microscopy and FRET, the CHC–IKK complexes were visualized together with NEMO or HSP90. The natural variability of protein amounts in unsynchronized A549 cells was used to obtain statistical correlation for several complexes, at natural levels and without invasive labeling. Analyses of voxel numbers indicated that: (i) CHC–IKK complexes are not part of the IKK signalosome itself but, likely, precursors of IKK–NEMO complexes. (ii) CHC–IKKβ complexes may arise from IKKβ–HSP90 complexes.
Clathrin forms complexes with IKKa, IKKb, and NEMO, but apparently not the canonical signalosome. These complexes are identified, for the first time, by affinity proteomics and triple FRET without altering molecular structure.
COVID-19 predisposes patients to a prothrombotic state with demonstrated microvascular involvement. The degree of hypercoagulability appears to correlate with outcomes; however, optimal criteria to ...assess for the highest-risk patients for thrombotic events remain unclear; we hypothesized that deranged thromboelastography measurements of coagulation would correlate with thromboembolic events.
Patients admitted to an ICU with COVID-19 diagnoses who had thromboelastography analyses performed were studied. Conventional coagulation assays, d-dimer levels, and viscoelastic measurements were analyzed using a receiver operating characteristic curve to predict thromboembolic outcomes and new-onset renal failure.
Forty-four patients with COVID-19 were included in the analysis. Derangements in coagulation laboratory values, including elevated d-dimer, fibrinogen, prothrombin time, and partial thromboplastin time, were confirmed; viscoelastic measurements showed an elevated maximum amplitude and low lysis of clot at 30 minutes. A complete lack of lysis of clot at 30 minutes was seen in 57% of patients and predicted venous thromboembolic events with an area under the receiver operating characteristic curve of 0.742 (p = 0.021). A d-dimer cutoff of 2,600 ng/mL predicted need for dialysis with an area under the receiver operating characteristic curve of 0.779 (p = 0.005). Overall, patients with no lysis of clot at 30 minutes and a d-dimer > 2,600 ng/mL had a venous thromboembolic event rate of 50% compared with 0% for patients with neither risk factor (p = 0.008), and had a hemodialysis rate of 80% compared with 14% (p = 0.004).
Fibrinolysis shutdown, as evidenced by elevated d-dimer and complete failure of clot lysis at 30 minutes on thromboelastography predicts thromboembolic events and need for hemodialysis in critically ill patients with COVID-19. Additional clinical trials are required to ascertain the need for early therapeutic anticoagulation or fibrinolytic therapy to address this state of fibrinolysis shutdown.
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BACKGROUNDProlonged cold ischemia is a risk factor for delayed graft function of kidney transplants, and is associated with caspase-3–mediated apoptotic tubular cell death. We hypothesized that ...treatment of tubular cells and donor kidneys during cold storage with a caspase inhibitor before transplant would reduce tubular cell apoptosis and improve kidney function after transplant.
METHODSMouse tubular cells were incubated with either dimethyl sulfoxide (DMSO) or Q-VD-OPh during cold storage in saline followed by rewarming in normal media. For in vivo studies, donor kidneys from C57BL/6 mice were perfused with cold saline, DMSO (vehicle), or QVD-OPh. Donor kidneys were then recovered, stored at 4°C for 60 minutes, and transplanted into syngeneic C57BL/6 recipients.
RESULTSTubular cells treated with a caspase inhibitor had significantly reduced capsase-3 protein expression, caspase-3 activity, and apoptotic cell death compared with saline or DMSO (vehicle) in a dose-dependent manner. Treatment of donor kidneys with a caspase inhibitor significantly reduced serum creatinine and resulted in significantly less tubular cell apoptosis, BBI, tubular injury, cast formation, and tubule lumen dilation compared with DMSO and saline-treated kidneys.
CONCLUSIONSCaspase inhibition resulted in decreased tubular cell apoptosis and improved renal function after transplantation. Caspase inhibition may be a useful strategy to prevent cold ischemic injury of donor renal grafts.
Hypertonic saline (HTS) has been used intravenously to reduce organ dysfunction following injury and as an inhaled therapy for cystic fibrosis lung disease. The role and mechanism of HTS inhibition ...was explored in the TNFα and IL-1β stimulation of pulmonary epithelial cells. Hyperosmolar (HOsm) media (400 mOsm) inhibited the production of select cytokines stimulated by TNFα and IL-1β at the level of mRNA translation, synthesis and release. In TNFα stimulated A549 cells, HOsm media inhibited I-κBα phosphorylation, NF-κB translocation into the nucleus and NF-κB nuclear binding. In IL-1β stimulated cells HOsm inhibited I-κBα phosphorylation without affecting NF-κB translocation or nuclear binding. Incubation in HOsm conditions inhibited both TNFα and IL-1β stimulated nuclear localization of interferon response factor 1 (IRF-1). Additional transcription factors such as AP-1, Erk-1/2, JNK and STAT-1 were unaffected by HOsm. HTS and sorbitol supplemented media produced comparable outcomes in all experiments, indicating that the effects of HTS were mediated by osmolarity, not by sodium. While not affecting MAPK modules discernibly in A549 cells, both HOsm conditions inhibit IRF-1 against TNFα or IL-1β, but inhibit p65 NF-kB translocation only against TNFα but not IL-1β. Thus, anti-inflammatory mechanisms of HTS/HOsm appear to disrupt cytokine signals at distinct intracellular steps.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
One of the cornerstone research models used in our laboratories is the induction of ischemic injury through cold ischemia followed by warm ischemia to donor kidneys to mimic the clinical realities of ...transplantation. The experimental design of the present study included bilateral nephrectomies on the day of syngeneic kidney transplant, with serum creatinine measured 24 hours postoperatively to measure acute function. Cold ischemia time in these experiments was always 30 minutes, and warm ischemia time was not standardized but always recorded. It became apparent that some transplanted kidneys that should have displayed injury were producing close to normal serum creatinine levels on postoperative day 1. In reviewing our data, we found a potential correlation between warm ischemia time and serum creatinine, in particular a significant proportion of low serum creatinine results (0.48 ± 0.26 mg/dL vs 1.99 ± 1.11 mg/dL; P < .05) was associated with warm ischemia times that were significantly shorter than our historical average (29.2 ± 2.7 min vs 35.7 ± 2.2 min; P < .05). The kidneys with lower serum creatinine also displayed lower apoptosis and brush border injury scores and fewer tubular casts. Therefore, we concluded that establishing a minimum warm ischemia time was just as important as standardized cold ischemia time to ensure consistent injury in this model.
•Consistent injury models are required to investigate intervention modalities.•Warm ischemia is a part of the ischemia-reperfusion injury cascade.•Warm ischemia and cold ischemia can be considered as separate events.
BACKGROUND.Prolonged cold ischemia (CI) is a risk factor for acute kidney injury after kidney transplantation. We endeavored to determine the pathways involved in the development of tubular cell ...injury and death before and after transplantation. We hypothesized that ex vivo cold storage before transplant would produce a different injury phenotype to that seen after engraftment in kidney transplants with or without CI.
METHODS.Four groups of mouse donor kidneys were studied(1) nontransplanted control kidneys; (2) donor kidneys subjected to ex vivo cold ischemia (CI); (3) donor kidneys subjected to kidney transplant without CI (Txp); and (4) donor kidneys subjected to CI followed by transplantation (CI+Txp).
RESULTS.Acute kidney injury only occurred in the CI+Txp group, which had significantly increased sCr versus the Txp group and the control mice. Histologically, the CI group demonstrated significantly increased tubular cell apoptosis and caspase-9 expression, whereas the Txp group demonstrated only mild brush border injury without apoptosis or necrosis. In contrast, the CI+Txp group had tubular cell apoptosis associated with expression of caspase-8, TNFR1, and increased serum TNF-α. CI+Txp also led to significantly higher ATN scores in association with increased RIP1, RIP3, pMLKL, and TLR4 expression.
CONCLUSIONS.Our results suggest distinct therapies are needed at different times during organ preservation and transplantation. Prevention of apoptosis during cold storage is best achieved by inhibiting intrinsic pathways. In contrast, prevention of cell death and innate immunity after CI+Txp requires inhibition of both the extrinsic death receptor pathway via TNFR1 and caspase-8 and inhibition of programmed necrosis via TLR4 and TNFR1.
AIM: To evaluate donation after circulatory death（DCD） orthotopic liver transplant outcomes hypoxic cholangiopathy（HC） and patient/graft survival and donor risk-conditions.METHODS: From 2003-2013, 45 ...DCD donor transplants were performed. Predonation physiologic data from UNOS Donor Net included preoperative systolic and diastolic blood pressure, heart rate, p H, SpO2, PaO2, FiO2, and hemoglobin. Mean arterial bloodpressure was computed from the systolic and diastolic blood pressures. Donor preoperative arterial O2 content was computed as hemoglobin（gm/d L） × 1.37（m L O2/gm） × SpO2%) +（0.003 × PaO2）. The amount of preoperative donor red blood cell transfusions given and vasopressor use during the intensive care unit stay were documented. Donors who were transfused ≥ 1 unit of red-cells or received ≥ 2 vasopressors in the preoperative period were categorized as the red-cell/multi-pressor group. Following withdrawal of life support, donor ischemia time was computed as the number-of-minutes from onset of diastolic blood pressure < 60 mm Hg until aortic cross clamping. Donor hypoxemia time was the number-of-minutes from onset of pulse oximetry < 80% until clamping. Donor hypoxia score was（ischemia time + hypoxemia time） ÷ donor preoperative hemoglobin.RESULTS: The 1, 3, and 5 year graft and patient survival rates were 83%, 77%, 60%; and 92%, 84%, and 72%, respectively. HC occurred in 49% with 16% requiring retransplant. HC occurred in donors with increased age（33.0 ± 10.6 years vs 25.6 ± 8.4 years, P = 0.014）, less preoperative multiple vasopressors or red-cell transfusion（9.5% vs 54.6%, P = 0.002）, lower preoperative hemoglobin（10.7 ± 2.2 gm/d L vs 12.3 ± 2.1 gm/d L, P = 0.017）, lower preoperative arterial oxygen content（14.8 ± 2.8 m L O2/100 m L blood vs 16.8 ± 3.3 m L O2/100 m L blood, P = 0.049）, greater hypoxia score >2.0（69.6% vs 25.0%, P = 0.006）, and increased preoperative mean arterial pressure（92.7 ± 16.2 mm Hg vs 83.8 ± 18.5 mm Hg, P = 0.10）. HC was independently associated with age, multi-pressor/redcell transfusion status, arterial oxygen content, hypoxia score, and mean arterial pressure（r2 = 0.6197）. The transplantation rate was greater for the later period with more liberal donor selection era 2（7.1/year）, compared to our early experience era 1（2.5/year）. HC occurred in 63.0% during era 2 and in 29.4% during era 1（P = 0.03）. Era 2 donors had longer times for extubation-to-asystole（14.4 ± 4.7 m vs 9.3 ± 4.5 m, P = 0.001）, ischemia（13.9 ± 5.9 m vs 9.7 ± 5.6 m, P = 0.03）, and hypoxemia（16.0 ± 5.1 m vs 11.1 ± 6.7 m, P = 0.013） and a higher hypoxia score > 2.0 rate（73.1% vs 28.6%, P = 0.006）.CONCLUSION: Easily measured donor indices, including a hypoxia score, provide an objective measure of DCD liver transplantation risk for recipient HC. Donor selection criteria influence HC rates.
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