Involvement of phosphatase and tensin homolog-induced putative kinase 1–Parkin-mediated mitophagy in septic acute kidney injury

Background:Studies have reported mitophagy activation in renal tubular epithelial cells(RTECs)in acute kidney injury(AKI).Phosphatase and tensin homolog-induced putative kinase 1(PINK1)and E3 ubiquitin-protein ligase Parkin are involved in mitophagy regulation;however,little is known about the role of PINK1-Parkin mitophagy in septic AKI.Here we investigated whether the PINK1-Parkin mitophagy pathway is involved in septic AKI and its effects on cell apoptosis in vitro and on renal functions in vivo.Methods:Mitophagy-related gene expression was determined using Western blot assay in human RTEC cell line HK-2 stimulated with bacterial lipopolysaccharide(LPS)and in RTECs from septic AKI rats induced by cecal ligation and perforation(CLP).Autophagy-related ultrastructural features in rat RTECs were observed using electron microscopy.Gain-and loss-of-function approaches were performed to investigate the role of the PINK1-Parkin pathway in HK-2 cell mitophagy.Autophagy activators and inhibitors were used to assess the effects of mitophagy modulation on cell apoptosis in vitro and on renal functions in vivo.Results:LPS stimulation could significantly induce LC3-II and BECN-1 protein expression(LC3-II:1.72±0.05 vs.1.00±0.05,P<0.05;BECN-1:5.33±0.57 vs.1.00±0.14,P<0.05)at 4 h in vitro.Similarly,LC3-II,and BECN-1 protein levels were significantly increased and peaked at 2 h after CLP(LC3-II:3.33±0.12 vs.1.03±0.15,P<0.05;BECN-1:1.57±0.26 vs.1.02±0.11,P<0.05)in vivo compared with those after sham operation.Mitochondrial deformation and mitolysosome-mediated mitochondria clearance were observed in RTECs from septic rats.PINK1 knockdown significantly attenuated LC3-II protein expression(1.35±0.21 vs.2.38±0.22,P<0.05),whereas PINK1 overexpression markedly enhanced LC3-II protein expression(2.07±0.21 vs.1.29±0.19,P<0.05)compared with LPS-stimulated HK-2 cells.LPS-induced proapoptotic protein expression remained unchanged in autophagy activator-treated HK-2 cells and was significantly attenuated in PINK1-overexpressing cells,but was remarkably upregulated in autophagy inhibitor-treated and in PINK1-depleted cells.Consistent results were observed in flow cytometric apoptosis assay and in renal function indicators in rats.Conclusion:PINK1-Parkin-mediated mitophagy might play a protective role in septic AKI,serving as a potential therapeutic target for septic AKI.

Diagnostic and Predictive Levels of Calcium-binding Protein A8 and Tumor Necrosis Factor Receptor-associated Factor 6 in Sepsis-associated Encephalopathy： A Prospective Observational Study

Background： Despite its high prevalence, morbidity, and mortality, sepsis-associated encephalopathy （SAE） is still poorly understood. The aim of this prospective and observational study was to investigate the clinical significance of calcium-binding protein A8 （S 100AS） in serum and tumor necrosis factor receptor-associated factor 6 （TRAF6） in peripheral blood mononuclear cells （PBMCs） in diagnosing SAE and predicting its prognosis. Methods： Data of septic patients were collected within 24 h after Intensive Care Unit admission fi-om July 2014 to March 2015. Healthy medical personnel served as the control group. SAE was defined as cerebral dysfhnction in the presence of sepsis that fulfilled the exclusion criteria. The biochemical indicators, Glasgow Coma Scale, Acute Physiology and Chronic Health Evaluation score II, TRAF6 in PBMC, serum S 100A8, S 10013, and neuron-specific enolase were evaluated in SAE patients afresh. TRAF6 and S 100A8 were also measured in the control group. Results： Of the 57 enrolled patients, 29 were diagnosed with SAE. The S 100A8 and TRAF6 concentrations in SAE patients were both significantly higher than that in no-encephalopathy （NE） patients, and higher in NE than that in controls （3.74 ± 3.13 vs. 1.08 ± 0.75 vs. 0.37 ± 0.14 ng/ml, P 〈 0.01 ; 3.18 ± 1.55 vs. 1.02 ± 0.63 vs. 0.47 ± 0.10, P 〈 0.01）. S 100A8 levels of 1.93 ng/ml were diagnostic of SAE with 92.90% specificity and 69.00% sensitivity in the receiver operating characteristic （ROC） curve, and the area under the curve was 0.86 （95% confidence interval [CI]： 0.76-0.95）. TRAF6-relative levels of 1.44 were diagnostic of SAE with 85.70% specificity and 86.20% sensitivity, and the area under the curve was 0.94 （95% CI： 0.88-0.99）. In addition, S 100A8 levels of 2.41 ng/ml predicted 28-day mortality of SAE with 90.00% specificity and 73.70% sensitivity in the ROC curve, and the area under the curve was 0.88. TRAF6 relative levels of 2.94 predicted 28-day mortality of SAE with 80.00% specificity and 68.40% sensitivity, and the area under the curve was 0.77. Compared with TRAF6, the specificity of serum S 100A8 in diagnosing SAE and predicting mortality was higher, although the sensitivity was low. In contrast, the TRAF6 had higher sensitivity for diagnosis. Conclusions： Peripheral blood levels of S 100A8 and TRAF6 in SAE patients were elevated and might be related to the severity of SAE and predict the outcome of SAE. The efficacy and specificity of S 100A8 for SAE diagnosis were superior, despite its weak sensitivity. S100A8 might be a better biomarker for diagnosis of SAE and predicting prognosis.

Ten Basic Principles about Critical Ultrasonography： Critical Care Practitioners Need to Know

With the noninvasive, convenient, and unique value in tile quick diagnosis and dynamic monitoring during the course of treatment, critical ultrasonography has been widely used in critical care medicine, regarded as the ＂visual stethoscope＂ and applied in medical education and training system. Based on years of experience in the application and promotion of critical ultrasonography, the authors of this study suggested that a proper understanding of critical ultrasonography is of most importance, and a consistent quality control system is required to minimize misdiagnosis,

Value of Kidney Disease Improving Global Outcomes Urine Output Criteria in Critically III Patients： A Secondary Analysis of a Multicenter Prospective Cohort Study

Background： Urine output （UO） is an essential criterion of the Kidney Disease Improving Global Outcomes （KD1GO） definition and classification system tbr acute kidney injury （AKI）, of which the diagnostic value has not been extensively studied. We aimed to determine whether AKI based on KDIGO UO criteria （KDtGOLro） could improve the diagnostic and prognostic accuracy, compared with KDIGO serum creatinine criteria （KDIGOscr）.Methods： We conducted a secondary analysis of the database of a previous study conducted by China Critical Care Clinical Trial Group （CCCCTG）, which was a 2-month prospective cohort study （July 1,2009 to August 31,2009） involving 3063 patients in 22 tertiary Intensive Care Units in Mainland of China. AKI was diagnosed and classified separately based on KDIGOt,o and KDlGOsc,. Hospital mortality of patients with more severe AKI classification based on KDIGOvo was compared with other patients by univariate and multivariate regression analyses. Results： The prevalence of AKl increased from 52.4% based on KDIGOscr to 55.4% based on KD1GOsc~ combined with KDIGOuo. KDIGOv~~ also restllted in an upgrade of AKI classification in 7.3% of patients, representing those with more severe AK1 classification based on KDIGOvo. Compared with non-AKI patients or those with maximum AKI classification by KDIGOscr, those with maximum AKI classification by KDIGOuo had a significantly higher hospital mortality of 58.4% （odds ratio [OR]： 7.580, 95% confidence interval [CI]： 4.141-13.873, P 〈 0.001）. In a multivariate logistic regression analysis, AKI based on KDIGOuo （OR： 2.891, 95% CI： 1.964-4.254, P 〈 0.001）, but not based on KDIGOscr （OR： 1.322, 95% CI： 0.902-1.939, P = 0.152）, was an independent risk factor for hospital mortality. Conclusion： UO was a criterion with additional value beyond creatinine criterion for AKI diagnosis and classification, which can help identify a group of patients with high risk of death.

Blocking Cyclic Adenosine Diphosphate Ribose-mediated Calcium Overload Attenuates Sepsis-induced Acute Lung Injury in Rats

Background： Acute lung injury （ALI） is a common complication of sepsis that is associated with high mortality, lntracellular Ca^2＋ overload plays an important role in the pathophysiology of sepsis-induced ALl, and cyclic adenosine diphosphate ribose （cADPR） is an important regulator of intracellular Ca^2＋ mobilization. The cluster of differentiation 38 （CD38）/cADPR pathway has been found to play roles in multiple inflammatory processes but its role in sepsis-induced ALl is still unknown. This study aimed to investigate whether the CD38/cADPR signaling pathway is activated in sepsis-induced ALl and whether blocking cADPR-mediated calcium overload attenuates ALl. Methods： Septic rat models were established by cecal ligation and puncture （CLP）. Rats were divided into the sham group, the CLP group, and the CLP＋ 8-bromo-cyclic adenosine diphosphate ribose （8-Br-cADPR） group. Nicotinamide adenine dinucleotide （NAD＋）, cADPR, CD38, and intracellular Ca^2＋ levels in the lung tissues were measured at 6, 12, 24, and 48 h after CLP surgery. Lung histologic injury, tumor necrosis factor （TNF）-a, malondialdehyde （MDA） levels, and superoxide dismutase （SOD） activities were measured. Results： NAD＋, cADPR, CD38, and intracellular Ca-＋ levels in the lungs of septic rats increased significantly at 24 h after CLP surgery. Treatment with 8-Br-cADPR, a specific inhibitor of cADPR, significantly reduced intracellular Ca^2＋ levels （P = 0.007）, attenuated lung histological injury （P = 0.023）, reduced TNF-a and MDA levels （P 〈 0.001 and P = 0.002, respectively） and recovered SOD activity （P = 0.031） in the lungs of septic rats. Conclusions： The CD38/cADPR pathway is activated in the lungs of septic rats, and blocking cADPR-mediated calcium overload with 8-Br-cADPR protects against sepsis-induced ALl.