As we all know, curative resection remains the only effective treatment for hepatocellular cancer (HCC). However, systemic inflammatory response syndrome always correlates with surgery, which may impose an impact on the clinical outcome of HCC patients who had undergone curative treatment. The present study is aimed at exploring the correlation between perioperative inflammatory mediators and recurrence risk of HCC.  This study retrospectively included 157 histologically confirmed single HCC patients (88 patients developed HCC again) who had received radical hepatectomy between January 2016 and May 2018 at the Department of Hepatobiliary Surgery, the People's Liberation Army General Hospital (PLAGH), China. The cut-off values for predicting recurrence were determined by receiver operating characteristic (ROC) curve analysis with estimation of the Youden index. Recurrence-free survival (RFS) was assessed using the Kaplan-Meier method, and the difference was compared between groups by the log-rank test. Univariate/multivariate analysis was performed to identify independent risk factors of postoperative tumor recurrence. α levels showed significant differences between groups. High preoperative serum IL6, IL8, and TNF-α levels were significantly associated with shorter RFS. Multivariate analysis revealed that preoperative serum IL6 > 8.45 pg/ml, preoperative serum IL8 > 68 pg/ml, preoperative serum TNF − α > 14.9 pg/ml, microvascular invasion (MVI), and maximum tumor size > 6 cm were independent predictors of RFS.  The perioperative serum IL1, IL2, and IL10 levels showed no difference between groups, whereas the serum IL6, IL8, and TNF-α levels were distinctly correlated with the postoperative tumor recurrence risk of HCC patients. The present study confirmed that high preoperative serum IL6, IL8, and TNF- Liver cancer is the sixth most common cancer and the fourth leading cause of cancer death worldwide, and hepatocellular cancer (HCC) accounts for 75-82% of the cases . In the α (TNF-α) also played some important roles during the evolvement of cancers [The chronic inflammatory response may play a crucial part in the evolvement of cancer, and inflammatory mediators are prominent players in these physiological processes , 3. Prev cancers , 19. Mor cancers . Further cancers . Based oIn the present study, we aim to explore the dynamic changes of perioperative inflammatory mediators and define the relationship between inflammatory markers and the prognosis of HCC patients.We retrospectively included 157 histologically confirmed single HCC patients (88 patients developed HCC again) who had received radical hepatectomy between January 2016 and May 2018 at the Department of Hepatobiliary Surgery, PLAGH, China, and this study was authorized by the PLAGH medical ethics committee.The entry criteria are as follows: age 18 years or older, histologically confirmed single HCC, Child-Pugh class A or class B that can reverse to class A after conventional therapy, had accepted radical hepatectomy, and had complete data of perioperative inflammatory mediators. Exclusion criteria included other malignancies other than HCC, nonsurgical interventions, and lack of follow-up.α concentrations were quantified by the IMMULITE® 1000 Immunoassay System  at preoperative and postoperative days 1, 3, 5, and 7. All data was retrospectively collected from original medical records at our hospital.We collected the following preoperative data: demographics and laboratory examinations. Operative parameters included operative time, operative procedure, Pringle maneuver, and total blood loss. According to the histopathologic reports, we determined the tumor number and size, tumor differentiation, microvascular invasion (MVI), and cirrhotic change. The serum IL1, IL2, IL6, IL8, IL10, and TNF-All patients had received radical hepatectomy by the same surgical team and received regular follow-up  in the outpatient department by the surgeon. During every follow-up, all patients received laboratory examination , alanine transaminase (ALT), and aspartase transaminase (AST)) and contrast-enhanced MRI examination. Follow-up data were obtained from our prospective database and through direct contact with patients and their families, and the follow-up deadline was December 2018.The present study is aimed at investigating the correlation between preoperative inflammatory mediators and the postoperative tumor recurrence risk of HCC.The secondary objective includes investigating the change of perioperative inflammatory mediators' level and identifying the independent predictors of recurrence-free survival  or deadline (December 2018) was calculated as RFS).U test or Student's t-test. Categorical data were summarized by frequency and were compared using the χ2 test or Fisher's exact test. The correlations between preoperative inflammatory mediators and maximum tumor size were analyzed by Pearson r. The RFS curves were plotted using the Kaplan-Meier method and compared by the log-rank test. The risk factors of postoperative tumor recurrence were determined by the univariate/multivariate Cox proportional hazard model. The optimal cut-off values for predicting recurrence were determined by ROC curve analysis with estimation of the Youden index. Significance was represented by p < 0.05, two sides. All statistical tests were done by SPSS 22.0 software.Continuous variables were described as the “mean ± standard deviation (SD)” or median (interquartile range), appropriately, and were compared using the Mann–Whitney This study retrospectively included 157 histologically confirmed single HCC patients who received curative hepatectomy from January 2016 to May 2018. During the follow-up, 88 patients developed HCC again. The median follow-up period for the recurrence group or nonrecurrence group was 24.0 (16.0 to 30.0) and 24.0 (17.0 to 27.0) months, respectively.p < 0.001), and MVI was more common in the recurrence group.α levels was shown in α concentrations of the recurrence group were significantly higher than those of the nonrecurrence group , but the serum IL1, IL2, and IL10 concentrations were comparable between groups . At postoperative day 1 and day 3, the serum IL6, IL8, and TNF-α concentrations of the recurrence group remained higher than those of the nonrecurrence group , whereas the serum IL1, IL2, and IL10 levels remained similar between groups . Five days after the operation, only serum TNF-α concentration was significantly different between groups , the serum IL1, IL2, IL6, IL8, and IL10 concentrations were comparable between groups . At the postoperative day 7, no significant difference was found in the serum IL1, IL2, IL6, IL8, IL10, and TNF-α concentrations between groups .The postoperative change of serum IL1, IL2, IL6, IL8, IL10, and TNF-r. Pearson r analysis revealed that preoperative serum IL6, IL8, and TNF-α levels were not associated with maximum tumor size ; furthermore, there was no relationship between preoperative serum IL6, IL8, and TNF-α levels and MVI status .As shown in α levels. ROC curve analysis  found that the optimal preoperative serum IL6, IL8, and TNF-α level cut-off values for the presence of recurrence were 8.45, 68, and 14.9, respectively  or high  preoperative IL6, groups with low  or high  preoperative IL8, and groups with low  or high  preoperative TNF-α. High preoperative serum IL6, IL8, and TNF-α levels were distinctly correlated with shorter RFS  after radical hepatectomy , preoperative serum IL8 > 68 pg/ml , preoperative serum TNF − α > 14.9 pg/ml , MVI , and maximum tumor size > 6 cm  were independent risk factors of recurrence-free survival in HCC patients  and preoperative serum IL6, IL8, and TNF-atectomy . Multivapatients .α levels were significantly correlated with the postoperative tumor recurrence risk of the HCC patients, and both univariate and multivariate analyses showed that preoperative serum IL6 > 8.45 pg/ml, preoperative serum IL8 > 68 pg/ml, preoperative serum TNF − α > 14.9 pg/ml, MVI, and maximum tumor size > 6 cm were independent predictors of recurrence-free survival. Furthermore, whether in patients with MVI or without MVI, or in patients with maximum tumor size > 5 cm or ≤5 cm, high preoperative serum IL6, IL8, and TNF-α levels were distinctly correlated with shorter RFS. Therefore, we concluded that the preoperative serum IL-6, IL8, and TNF-α levels may serve as potential predictors for the clinical outcome of HCC patients.Our results showed that preoperative serum IL6, IL8, and TNF-κB, the signal transducer and activator of transcription. Furthermore, Porta et al. [It is well known that inflammation and immune mediators play an important role in cancer progression , 3. Casaa et al.  showed ta et al.  concludeα levels were significantly correlated with RFS. The expression of IL-8 was increased in cancer cells and tumor-associated macrophages, suggesting that IL-8 may play a crucial role in the tumor microenvironment. Clinically, some studies showed that IL8 was involved in the evolvement of cancer. For example, Welling et al. [α and the development of liver cancer [α levels may serve as predictors for the tumor recurrence risk of HCC patients.In addition to IL6, we also found that high preoperative serum IL8 and TNF-g et al.  concludeg et al.  showed tg et al. . There ar cancer . Altogetα) may be associated with liver fibrosis and cirrhosis. As shown in previous studies, Xiang et al. [α) levels in cirrhosis patients were significantly higher than those in healthy controls. Furthermore, D'Anzeo et al. demonstrated that various aberrantly expressed miRNAs  are correlated with the prognosis of HCC patients. Therefore, further study is needed to explore the signaling pathway that connects inflammatory mediators with miRNAs, and targeting those pathways may contribute to the treatment of HCC [Preoperative serum inflammatory mediators  and the prognosis of patients with HCC, few studies examined these cytokines at the same time. Differently, the present study strictly collected data of perioperative inflammatory mediators, including IL1, IL2, IL6, IL8, IL10, and TNF-α should not be used as new predictors until large prospective studies can be performed. Last, a previous study showed that IL6 was not related to the clinical outcome in patients with resectable hepatocellular carcinoma [Our study has several limitations. First, it is a retrospective study, so we cannot avoid some potential bias. Second, this study only included patients from one centre, and its sample is relatively small, so IL6, IL-8, and TNF-arcinoma , so our α levels were significantly correlated with the postoperative tumor recurrence risk of the HCC patients. Therefore, we concluded that preoperative sera IL6, IL8, and TNF-α may predict the postoperative recurrence of hepatocellular carcinoma.Our findings show that high preoperative serum IL6, IL8, and TNF-


Candidatus Nitrospira defluvii and nitrite oxidizing bacteria Nitrosomonas sp. Nm47, while that on denitrification performance in SPDB is mainly embodied in the change of composition and amount of solid carbon substrate degrading denitrifying bacteria Pseudomonas sp., Myxobacterium AT3-03 and heterotrophic denitrifying bacteria Dechloromonas agitate, Thauera aminoaromatica, Comamonas granuli and Rubrivivax gelatinosus.In order to solve the problems of high energy consumption, complex process and low nitrogen removal efficiency in the currently available low carbon source wastewater treatment processes, a novel coagulation sedimentation/post-solid-phase denitrification biofilter process (CS-BAF-SPDB) was proposed. The effect of temperature on the nitrogen removal performance of BAF-SPDB was intensively studied, and the mechanism of the effect of temperature on nitrogen removal performance was analyzed from the perspective of microbial community structure by using the polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). The results showed that, to realize favorable nitrifying and denitrifying performance simultaneously in the BAF-SPDB unit, the operation temperature should be set above 18 °C. In addition, the influence of the macro operation parameters on the performance of the BAF and SPDB has a direct relationship with the dynamic changes of the micro microbial community. The influence of temperature on nitrification performance in BAF was mainly embodied in the change of composition, amount and activity of ammonia oxidizing bacteria  In recent years, municipal domestic wastewater has become characterized by a low carbon to nitrogen (C/N) ratio . Several2. Considering the biodegradability, the start-up cycle and the stability of the degradation process, we have chosen the biodegradable polymer polycaprolactone (PCL) instead of other biodegradable polymers in SPDB [In this study, we propose a novel post denitrification strategy based on solid-phase denitrification to realize high nitrogen removal efficiency from wastewater with a low C/N ratio. This strategy is termed coagulation sedimentation-post solid-phase denitrification biofilter process (CS-BAF-SPDB). In the first step, most of the suspended solids and organic matter are removed in the coagulation sedimentation unit (CS), which could not only ease the blockage of the subsequent biological filters unit, but also to avoid the impact of organic matter on the nitrification. In the second step, ammonia nitrogen is oxidized to nitrate nitrogen in the biological aeration filter (BAF). Compared with the activated sludge process, the advantage of BAF is that the biomass of nitrifying microorganism is guaranteed, and sludge bulking would not occur even under low organic carbon loading and low dissolved oxygen. The effluent of BAF then flows into the solid-phase denitrification biofilter (SPDB), and the nitrate nitrogen in the effluent is converted to N in SPDB ,10,11,12 in SPDB . In prev in SPDB ,10,11,12Temperature is one of the key factors affecting nitrogen removal efficiency of BAF-SPDB process ,15. It nThe start-up of this novel process and the effect of influent carbon/nitrogen ratio, gas/water ratio, hydraulic detention time and influent ammonia loading on the nitrogen removal efficiency of BAF-SPDB was investigated in the preliminary study in our laboratory . On thisPolycaprolactone (PCL) and clay ceramsite carriers were kindly supplied by Shenzhen Esun Industrial Co. Ltd and Jiangxi Pingxiang Sanhe Ceramics Co., Ltd. Their physical–chemical properties are shown in Cr) = 148–185 mg/L, ρ(NH4+-N) = 26.7–56.8 mg/L, ρ(TN) = 28.6–70.2 mg/L, ρ(SS) = 140.0–152.5 mg/L. To investigate the effect of operation conditions on nitrogen removal efficiency of the process, CODCr, NH4+-N and PO4−-P concentration in the feed water were controlled by dilution or adding sodium acetate ammonium chloride and potassium dihydrogen phosphate to maintain the stability of water quality.Domestic wastewater from student dormitories of Chongqing University, China  was used as feed water. The water quality parameters of the influent were as follows: ρ, was used as seed source for BAF and SPDB, and the concentration of the activated sludge was 4.2 g/L and 5.6 g/L, respectively.Activated sludge, obtained from the aerobic and anaerobic tank of AThe start-up of the CS-BAF-SPDB process was successfully achieved in the previous study , and theGenomic DNA samples were extracted by using rapid extraction kit of silt genomic DNA .Taking sample genomic DNA as templates, hypervariable region sequences of samples of 16S rDNA were amplified using two universal primers: GC-338F and 518R bacterial. System for PCR amplification (50 μL): 10 × PCR buffer 5 μL, dNTP (2.5 mM) 3.2 μL, rTaq (5 U/μL) 0.4 μL, GC-338F20 μM1 μL, 518R (20 μM) 1 μL, template DNA 50 ng, adding ddH2O to 50 μL.Program PCR amplification: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 1 min, annealing at 55 °C for 45 s, extension at 72 °C for 1 min, 30 cycles, and final extension at 72 °C for 10 min.PCR products were purified and recycled using DNA Gel Extraction Kit of OMEGA.PCR Instrument was T-gradient produced by Biometra, Gel Imager was Gel-Doc2000 produced by Bio-Rad. The universal primer information is shown in 10 μL PCR products were taken for DGGE analysis. Electrophoresis was proceeded at 150 V, 60 °C, in 1 × TAE buffer for 5 hours with a denatured gradient from 35% to 55%, and 8% polyacrylamide gel. The chemical denaturing agent was 100% urea 7 mol/ L, along with 40% acrylamide(v/v). The level of DGGE gel formulations  were amo2H5OH and 2.5 mL CH3COOH).Fixed for 15 min with fixative .Stained for 15 min with silver dye (a constant volume of 500 mL with 1 g AgNOCleaned with Milli-Q pure water for 20 s and 2 min each time.Colored for 5–7 min with color solution (a constant volume of 500 mL with 7.5 g NaOH and 2.5 mL 37% HCHO).2H5OH and 2.5 mL CH3COOH).Finally, the reaction was terminated with stop solution (a constant volume of 500 mL with 50 mL CAfter denaturing gradient gel electrophoresis (DGGE), the steps of silver staining were interpreted as follows:DGGE strips were cut and recovered with sterilized scalpel, and recycled target band using Poly-Gel DNA Extraction Kit of OMEGA. 2 μL recovered products were used as template, using 338 F/518 R as primers to PCR reactions.2O to 50 μL.PCR amplification system (50 μL):10 × PCR buffer 5 μL, dNTP (2.5 mM) 3.2 μL, rTaq (5 U/μL) 0.4 μL, 338F (20 mM) 1 μL, 518R (20 mM) 1 μL, template DNA 1 μL, adding ddHPCR amplification program: pre-denaturation at 94 °C for 4 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 30 s, 30 cycles, and final extension at 72 °C for 10 min.The re-amplified DNA fragments were recovered, purified and connected to the carrierpMD18-vectors. Then they were transformed into DH5α competent cells, and positive clones were screened. Finally, bacterial 16S rDNA fragments inserted in broth were sequenced by BGI.The dominant DGGE bands in the lane of BAF and SPDB, which changed obviously during the temperature changing process, were successfully re-amplified, cloned, sequenced and aligned with BLAST to get access to the 16S rDNA sequence of the most similar to type strain with BLAST program for homology in GenBank. Phylogenetic tree was constructed with the neighbor-joining method by employing MEGA5 software, bootstrap index was 1000.3−-N in the effluent of BAF showed almost no change. However, nitrification in BAF was obviously inhibited with the temperature decreasing from 18 °C to 13 °C, and NH4+-N removal efficiency decreased from 98.0% at 18 °C to 78.1% at 13 °C  was a common denitrifying bacterium in solid-phase denitrification process [Comamonas granuli had poor ability on cold tolerance, and therefore can only survive in a certain temperature range. DGGE band 12, on behalf of Rubrivivax gelatinosus, represented the predominant strain, the biomass of which changed very little during the temperature changing process, suggesting that the microorganism has stronger resistance to low temperature. As can be seen from Acinetobacter sp. (band 11) and Bacillus sp. (band 13) [Comamonas Granuli (band 8) disappeared from the strip on denitrification was therefore compensated, the further biomass decrease of polymers degrading denitrifying bacteria Myxobacterium AT3-03 significantly reduced the organic carbon release. Due to the insufficient electronic donors supply, denitrification in SPDB was greatly inhibited. As can be seen from atinosus ,33,34,35fication ,36. Withfication  also fou process . The briband 13) ,39 graduAcinetobacter sp. and Bacillus sp., two rod-shaded bacteria, gradually developed into dominant bacteria when the temperature decreased to 13 °C. To track the biofilm development on the clay ceramsite and PCL carriers and elucidate the effect of temperature on biofilm structure, ESEM observations were conducted under the temperature of 26 °C, 20 °C, 18 °C and 13 °C. 4+-N and TN removal efficiency of BAF-SPDB unit can reach more than 96.2% and 81.2% when the temperature is above 18 °C.In the CS-BAF-SPDB combined process, to realize favorable nitrifying and denitrifying performance simultaneously in the BAF-SPDB unit, the temperature should be controlled above 18 °C when the influent C/N ratio and gas/water ratio of BAF is 3:1 and 4:1, respectively, and HRT of BAF and SPDB is 3.5 h and 1.5 h, respectively. The NHCandidatus Nitrospira defluvii and nitrite oxidizing bacteria Nitrosomonas sp. Denitrification performance of Nm47 in SPDB is mainly embodied in the change of composition and amount of solid carbon substrate degrading denitrifying bacteria Pseudomonas sp., Myxobacterium AT3-03 and heterotrophic denitrifying bacteria Dechloromonas agitate, Thauera aminoaromatica, Comamonas granuli and Rubrivivax gelatinosus. Meanwhile, the change of microbial community structure of heterotrophic nitrification and aerobic denitrification with operation conditions also plays a very important role in ensuring the stable nitrification and denitrification under low temperature.In addition, the influence of the macro operation parameters on the performance of the BAF and SPDB has a direct relationship with the dynamic changes of the micro microbial community. The influence of temperature on nitrification performance in BAF is mainly embodied in the change of composition, amount and activity of ammonia oxidizing bacteria


Currents through these channels (ITRPM7) are robustly potentiated when the cell interior is exchanged with low Mg2+-containing buffers. ITRPM7 is also potentiated by phosphatidyl inositol bisphosphate P2) and suppressed by its hydrolysis. Here we characterized internal Mg2+- and pH-mediated inhibition of TRPM7 channels in HEK293 cells overexpressing WT voltage-sensing phospholipid phosphatase (VSP) or its catalytically inactive variant VSP-C363S. VSP-mediated depletion of membrane phosphoinositides significantly increased channel sensitivity to Mg2+ and pH. Proton concentrations that were too low to inhibit ITRPM7 when the VSP-C363S variant was expressed (pH 8.2) became inhibitory in WT VSP–expressing cells. At pH 6.5, protons inhibited ITRPM7 both in WT and VSP C363S–expressing cells but with a faster time course in the WT VSP–expressing cells. Inhibition by 150 μm Mg2+ was also significantly faster in the WT VSP–expressing cells. Cellular PIP2 depletion increased the sensitivity of TRPM7 channels to the inhibitor 2-aminoethyl diphenyl borinate, which acidifies the cytosol. Single substitutions at Ser-1107 of TRPM7, reducing its sensitivity to Mg2+, also decreased its inhibition by spermine and acidic pH. Furthermore, these channel variants were markedly less sensitive to VSP-mediated PIP2 depletion than the WT. We conclude that the internal Mg2+-, polyamine-, and pH-mediated inhibition of TRPM7 channels is not direct but, rather, reflects electrostatic screening and resultant disruption of PIP2–channel interactions.Transient receptor potential cation channel subfamily M member 7 (TRPM7) is an ion channel/protein kinase belonging to the TRP melastatin and eEF2 kinase families. Under physiological conditions, most native TRPM7 channels are inhibited by cytoplasmic Mg Biphasichannels . By contchannels . The mec fashion . The pol fashion , are alsloses it –27. In sh the WT .2+ and polyamines applied on the extracellular side produce a rapid voltage-dependent channel pore block  conductance, seen only with higher Mg2+ concentrations (>100 μm) . We hypothesized that, in whole-cell recordings, current rundown in the presence of Mg2+ reflects a gradual increase in the channels' sensitivity to Mg2+, akin to the sensitization observed in cell-free patches  . In some patches . Currentes e.g. , 32) and and2+ isholipids . Presumaholipids –36. Rundolutions .2 (hereafter referred to as PIP2) by phospholipase C β and γ inhibits whereas exogenous PIP2 activates TRPM7 channels Pchannels , 34. Expchannels , 38, supchannels .2+, polyamines, and acidic pH represents screening (electrostatic shielding) of negative charges on the phospholipid co-factors of these channels without directly demonstrating this , an inhibitor that acidifies the cytosol  were significantly less sensitive to PIP2 depletion than WT channels. These observations revealed that inhibition by internal Mg2+ and other cations shares a common mechanism and depends on cellular PIP2 levels.We proposed previously that inhibition by high internal Mging this . Here we cytosol , is alsoSer-1107  mutants,2 depletion on endogenous TRPM7 channel activity. We compared TRPM7 channel currents in HEK cells transfected with WT (active) and C363S mutant (inactive) CiVSP . In GFP+ cells transfected with C363S, there was no noticeable current reduction over time with 10 μm Mg2+ (A), whereas in WT VSP–expressing cells, the current declined during the experiment (B and C).HEK293 cells express significant magnesium-inhibited cation currents representing TRPM7 channel activity , 30. We e) CiVSP , 42. Fig μm Mg2+ A, whereaperiment , B and Cm [Mg2+], by contrast, was sufficient to inhibit TRPM7 channels in all tested WT cells and the majority of C363S-expressing cells (D–F). Time courses for 10 μm and 150 μm internal Mg2+ are shown in C and F; in the absence of PIP2 depletion (i.e. C363S expression), currents developed slowly after the whole-cell configuration was established and usually reached a maximum 2–6 min later .150 μng cells , D–F. Tiunctions , C and F2+-induced current decay that follows the initial rise phase , TRPM7 currents did not decay noticeably at 10 μm Mg2+ during our recordings. By contrast, expression of WT VSP and consequent PIP2 depletion resulted in a drastic sensitization of the channels to Mg2+; ∼50.8% to 61.1% of the maximum current declined after 6 min of dialysis. WT VSP–expressing cells that showed less decay most likely represent a subpopulation where the enzyme activity was low, leaving PIP2 levels high . Alternatively, these cells may express high levels of endogenous PI 5-kinase enzymes thought to be responsible for PIP2 replenishment . The decrease was more pronounced in WT VSP–expressing cells; on average, 78.9% of the current compared with 34.4% for C363S (B and C). In a minority of C363S-expressing cells, 150 μm was ineffective, producing no current reduction, whereas such an Mg2+-insensitive population was absent among WT VSP–expressing cells (B). In WT VSP–expressing cells, percent remaining current was indeed higher at 10 μm  than 150 μm Mg2+ , suggesting that current decay is faster at higher Mg2+ concentrations.The higher 150 μng cells B. The deor C363S , B and Cng cells B. In WT at 10 μm D, black  μm Mg2+ D, red, sA). This variability may be due to the heterogeneity of HEK cell resting membrane potentials and consequent differences in basal lipid phosphatase activity during culturing (see “Discussion”). Because culturing, transfection, and voltage protocols were identical for all cells, it is unlikely that the degree of VSP activation during the recording was the cause of the observed disparity . The decay time constant (τ) was significantly lower with increased internal Mg2+ concentration (E and F). Thus, at increased Mg2+ concentrations, the apparent onset of inhibition was accelerated in PIP2-depleted cells.We next compared the decay phases of the current in WT VSP–expressing cells for both Mgntration , C and Fntration , E and Fn = 12 cells) and C363S-expressing cells (n = 8 cells) were compared for 150 μm Mg2+ but were not statistically significantly different .Exponential decay time constants for WT  but only ∼2 min in PIP2-depleted (WT) cells, a 3-fold change (G and I). At 150 μm [Mg2+], the times to reach maximum were decreased in both C363S- and WT-expressing cells (H). This suggested that the effect of increasing [Mg2+] on the time to reach maximum current can also be mimicked by PIP2 depletion. The time to reach maximum current in each cell is set by a competition between the rising and the decay phase; i.e. when inhibition is accelerated, it will overwhelm the rising phase and manifest as an earlier drop in current magnitude.We next focused our attention on the rising phase of TRPM7 current and its dependence on PIPd change , G and Ing cells H. This s2-depleted cells reflected a faster current activation, we fitted the rise times with monoexponential functions for C363S- and WT VSP–expressing cells. We limited our analysis to WT VSP–expressing cells that showed decay and C363S-expressing cells that showed no decay in current. At 10 μm Mg2+, the time constants were 94.01 ± 19.03 for C363S and 72.9 ± 12.36 for WT VSP (mean ± S.E.). Thus, there was a tendency for PIP2 depletion to accelerate the initial rise of current, which was not statistically significant (Student's t test). For 150 μm, such a comparison was not performed because the current did not increase in most WT VSP–expressing cells (G–I) is primarily due to an effect on the decay phase rather than the initial potentiation of the current.To determine whether the shorter times to maximum in PIPng cells . We concng cells , G–I is 2 levels, as hypothesized previously (+] ≈ 10 nm) did not suppress TRPM7 channels in control C363S cells but became inhibitory in WT VSP–transfected cells (+] ∼ 1 μm) (A). In WT VSP–expressing PIP2-depleted cells, the current reduction at pH 6.5 was markedly faster (A and B). The internal free Mg2+ concentration in these experiments was held constant at 100 nm, a low concentration that prevents channel rundown (B and C).Next we investigated whether the dependence of native TRPM7 channel activity on cytosolic pH is also governed by cellular PIPeviously . We tested cells  20). In. In2 lev ∼ 1 μm) A. In WT y faster , A and B rundown , 40. 2 m rundown , B and CB) showed a significant interaction between PIP2 depletion and the inhibitory effect of protons (p < 0.0001). Thus, decreasing the cytoplasmic pH had a higher inhibitory effect on TRPM7 channels that were PIP2-depleted (i.e. WT VSP–expressing cells) compared with nondepleted channels (C363S VSP).A two-way analysis of variance performed using TRPM7 current amplitudes remaining 2 min after reaching maximum B showed 2+. In the perforated patch, TRPM7 channels do not run down, presumably because PIP2 is not depleted. We transfected HEK cells with mTRPM7 and studied its dependence on acidification induced by application of sodium propionate, a salt of a weak acid . In this case, repeated application of 20 mm propionate resulted in progressively stronger inhibition of the current. 3 μm Mg2+ is sufficient to support TRPM7 current rundown, likely involving the high-affinity Mg2+ inhibitor site (m) Mg2+ concentration, which does not support rundown, repeated applications of propionate inhibited the current to a similar extent . This behavior was similar to that seen in the perforated patch mode . Therefore, PIP2 depletion in whole-cell recordings results in stronger inhibition by cytoplasmic protons, which is not seen in perforated-patch recordings when PIP2 is not depleted. This is in agreement with the experiments described in 2 was depleted by VSP expression. Thus, the pH and Mg2+ sensitivities of TRPM7 channels are interdependent. The role of low micromolar Mg2+ in channel rundown only becomes apparent when PIP2 is depleted during whole-cell dialysis because the channels do not run down in the perforated patch even though the cytoplasmic Mg2+ is in the millimolar range.To examine the effect of cytoplasmic acidification on TRPM7 channels in the absence of rundown, we employed the perforated patch recording configuration, which does not perturb the cellular Mgm 2-APB in HEK cells to determine whether it depended on PIP2. In C363S CiVSP–expressing cells, this concentration of 2-APB had only a small effect on the current at internal free [Mg2+] of 400 nm (A and C). By contrast, in WT CiVSP–expressing cells, 2-APB robustly inhibited the current in 1–2 min (B and D). Washout of the drug fully reversed inhibition in C363S but only partially in some WT VSP–expressing cells (B and D). Current decay in WT VSP–expressing cells was substantially slower when 0.05% DMSO was applied without 2-APB (E). On average, 2-APB inhibited 81.8% of the current for WT versus 8% for C363S cells, quantified ∼3.0 min after application (E). 2-APB inhibition was also increased in cells expressing WT DrVSP, which activates at more depolarized potentials than Ciona enzyme (F). As expected, 2-APB inhibition was voltage-independent (A and B) . As seen with ramps reaching +85 mV (E). This observation is in agreement with reduced current magnitudes at break-in (I0) measured in WT VSP–expressing cells  and suggests that VSP is active and can deplete PIPs in HEK cells even without depolarizing voltages.TRPM7 and TRPM6 channel activity is commonly recorded by applying voltage ramps reaching +80 to +100 mV see  (6). To g +85 mV , native g +85 mV E. This o+ conditions. Elevating the external [K+] from 5.3 mm of the normal RPMI medium to 25.3 mm is expected to shift the K+ equilibrium potential by +41 mV. Break-in current amplitudes in WT VSP-transfected cells grown in low (15 cells) and high [K+] (10 cells) that exhibited current decay were not significantly different  (F). We observed no differences in VSP-induced current decay in cells grown in 5.3 or 25.3 mm [K+] either (data not shown). The mean Imax was higher for C363S than WT VSP–expressing cells at both K+ concentrations (p < 0.05) (F). These results suggest that either the HEK cell membrane is depolarized already or there is significant lipid phosphatase activity that does not require depolarization. To address this question, we measured the resting membrane potentials of HEK cells in perforated patch recordings, obtaining mean ± S.D. values of −59.7 ± 2.1 mV, −47.5 ± 2.7 mV, −34.8 ± 1.7 mV, −32.4 ± 1.2 mV, −33.9 ± 2.4 mV, and −27.8 ± 0.9 mV (6 cells), similar to published values. Cells with more hyperpolarized membrane potentials exhibited a prominent outward K+ current upon switching to voltage clamp (data not shown).HEK cells have reported membrane potentials of −40 to −50 mV . We ther = 0.05) F. We obs < 0.05) F. These Ciona ortholog (G). By contrast, currents did not decay in cells transfected with the inactive DrVSP C302S mutant (G). Collectively, the data presented in Fig. S1  is activated at more depolarized membrane potentials than its ortholog . The halortholog . Expressortholog G. By conS mutant G. Collecsee also  stronglyA) of TRPM7 and has been reported to make Mg2+-insensitive ion channels when substituted with glutamate (2+ (see “Introduction”), we tested various S1107 mutants for their sensitivity to spermine and pH. We first tested the S1107E mutant, originally described as Mg2+-insensitive (B–E). We further examined the 1107 position by mutagenizing this serine to positively charged lysine and arginine instead of negatively charged glutamate. Both lysine and arginine mutants also exhibited diminished Mg2+ sensitivity accompanied by reduced spermine and pH sensitivity (B–E). As a next step, we mutagenized the same serine to alanine, glutamine, threonine, and aspartate. Surprisingly, no correlation between the charge of this residue and channel sensitivity to Mg2+ was observed. However, we found that the size (surface area) of the substituting residue determined the channel phenotype. For example, changing Ser-1107 to glutamic acid or glutamine, having a similar size but differing in charge, both resulted in Mg2+-insensitive channels .Ser-1107 lies immediately outside of the TRP domain A of TRPMlutamate . This seensitive . Indeed,ensitive , B–E. Wesitivity , B–E. As2+ internal solution, whereas S1107E, S1107R, S1107K, and S1107Q currents were at their maximum magnitude at break-in (I0) and did not rise (B and D). On the other hand, WT, S1107A, and S1107T mutants showed only very small current rises in the presence of internal 1.7 mm Mg2+, 3 mm spermine, and pH 5.5 (C and D). This shows that the rising phase of TRPM7 current after break-in is due to the removal of inhibitory cations such as Mg2+ or polyamines and protons. Because S1107E, S1107R, S1107K, and S1107Q (gain-of-function (GOF) mutations) are insensitive to these inhibitory cations, they also do not show a rising phase with 0 internal Mg2+. The S1107C mutant failed to express substantial currents, whereas S1107D behaved as the other GOF mutants (data not shown). These experiments demonstrate that Mg2+ is not unique in its inhibition of TRPM7 and shares this property with other cations, such as polyamines and protons. Nevertheless, GOF mutations did not completely eliminate the Mg2+ sensitivity of TRPM7. At very high free Mg2+ concentrations (tested at 2.9 and 4.9 mm), both the S1107E and S1107K mutant channels were partially inhibited (data not shown).WT, S1107A, and S1107T TRPM7 magnitudes increased after break-in with 0 Mgnot rise , B and Dd pH 5.5 , C and D2+, spermine, and pH sensitivity (GOF mutants) by co-expressing CiVSP. We reasoned that if Mg2+, spermine, and protons inhibit TRPM7 by screening PIP2-negative charges, then depleting membrane PIP2 would mimic this behavior. In other words, Mg2+-insensitive mutants may also be VSP-insensitive. Fig. 8A shows whole-cell recordings of WT TRPM7 and two GOF mutants with 0 Mg2+i. As expected, the WT current in VSP-expressing cells increased (because of cytosolic Mg2+ washout) and then decayed over time because of further PIP2 depletion. By contrast, S1107E and S1107R currents lacked a rising phase and decayed significantly less over the same time period (A and B). This experiment demonstrated that channel sensitivity to Mg2+, polyamines, and protons strongly correlates with their sensitivity to PIP2 depletion, suggesting that they reflect the same process. To explore the sensitivity of TRPM7's local structure to changes at position 1107, a truncated model of TRPM7 was constructed and used to probe the consequences of changes at position 1107 . Replacement of serine with various residues examined here caused very modest solvent exposure changes, 1–4%, for residues within 8 Å of position 1107. This result was anticipated because the software we used finds only the structure's closest energy minimum and does not allow for the substantial structural changes necessary to locate a new global energy minimum. The degree of instability/stability introduced into the structure by each residue change was estimated by calculating the change in the force field free energy of the 1107 residue and neighboring residues (within 8 Å) relative to the native structure (Ser-1107). The results are shown in Fig. 8D , where the total free energy together with electrostatic and nonbonded components are plotted. The total and nonbonded free energies for the Gln , Lys , and Arg  substitutions are very large and unfavorable, indicating an extremely large driving force for structural change. Other substitutions (Asp and Glu) are associated with smaller free energy changes but are also unfavorable and of substantial magnitude, indicating a driving force for structural changes in these mutants. Last, the electrostatic interactions are relatively small in magnitude. These results suggest that with Ala, Ser, Cys, or Thr at position 1107, the structure is stable whereas it is not with Asp, Gln, Lys, or Arg at the same position.As a logical next step, we investigated the behavior of mutants with diminished Mg2+, spermine, and pH on the other. We employed expression of VSP to dephosphorylate the endogenous plasma membrane PIP2 and other phosphoinositide substrates, such as PIP3 (2+ and proton concentrations that were not inhibitory in inactive C363S VSP variant–expressing cells (10 μm Mg2+ and pH 8.2) significantly inhibited the native TRPM7 channel current when PIP2 was depleted by WT VSP activity. At higher concentrations, Mg2+ (150 μm) and protons (pH 6.5) were inhibitory in both WT- and C363S VSP–expressing cells but with a faster time course in the former. Native TRPM7 current inhibition by 2-APB was significantly more robust in WT Ciona or zebrafish VSP-expressing cells. Point mutations reducing TRPM7 channel sensitivity to Mg2+ also reduced its sensitivity to spermine and pH. Point mutants insensitive to these cations were also less sensitive to VSP co-expression.We have explored the relationship between well-known regulators of TRPM7 channels: membrane phosphoinositides on one hand and cytoplasmic Mg3,4,5)P3 . Our mai2+, the TRPM7 current develops slowly, reaching maximum amplitude after several minutes of cell dialysis (A–C). It is thought that this rise time reflects the removal of cytosolic Mg2+ (which likely occurs in seconds) and other downstream events. Whole-cell recordings (without VSP expression) with no Mg-ATP are accompanied by gradual PIP2 depletion (e.g. Refs. 2+ and ATP loss (A).Specifically, several parameters were changed by VSP expression: the extent of current reduction after the initial rise, measured at 5 or 6 min; the time constant of current decay; and the time to reach maximum. During whole-cell recording with low Mgdialysis , A–C. ItATP loss , heterolATP loss A.2+] in mammalian cells is in the 0.5–1 mm range (–2+ (and proton) removal from PIP2 molecules that are bound to the channels in an intact cell because the average pKa of PIP2 is estimated to be near pH 6.3 , supporting the view that PIP2 is a required cofactor for proper TRPM7 channel function.Stimulation of Gαo effect . Applica reports , 34. In 2 dependence within the physiological range as opposed to applying exogenous water-soluble PIP2 analogs at very high concentrations. In some cases, exogenously applied PIP2 activated ion channels that were later found to be PIP2-insensitive when endogenous phospholipids were depleted by using genetically engineered tools . Surprisingly, even without applying long depolarizing steps, which VSP is believed to require for activation . For HEK cells expressing WT and C363S, the break-in and maximum current amplitudes in high and normal K+ (F) were not statistically significantly different (Student's t test). Our own measurements of HEK cell membrane potential in 4.5 mm [K+]o showed that it varied between −59 and −27 mV (see “Results”). This points to the existence of substantial VSP activity at these negative membrane potentials. Accordingly, it has been demonstrated previously that the CiVSP enzyme is active over a wide range of membrane potentials achievable in patch clamp experiments  and may, by itself, be sufficient to activate VSP. This has been proposed for a similar voltage protocol (−120 to +100 mV applied at 1 Hz) used to record TRPM6 .We also looked for acute effects of VSP on TRPM7 channel currents by applying a long (2 s) depolarizing step either to +100 mV or to −10 mV and comparing the speed of current decay but saw no noticeable acceleration of current inhibition with +100 mV steps, a sufficient depolarization for VSP activation, used by other investigators for this purpose P3 while generating PI(4)P in the process  located in the TRP domain (A) to neutral glutamines resulted in nonfunctional channels : substitution with negatively charged glutamate resulted in channels with greatly reduced Mg2+ sensitivity. We investigated what other amino acid substitutions in this position would reduce Mg2+ sensitivity (gain-of-function mutations). Surprisingly, substituting Ser-1107 with positively charged arginine or lysine resulted in an identical phenotype (8D). Uncharged glutamine also gave rise to channels insensitive to Mg2+ (8D). Substitution with alanine or threonine, on the other hand, resulted in channels that behaved like the WT. We then tested whether these point mutants were sensitive to other positively charged inhibitors such as spermine and protons, finding that the GOF mutants were strikingly less sensitive to both, whereas the S1107A and S1107T mutants were inhibited by all three, like the WT channel . In combination, these results suggest that Ser-1107 is critical for the sensitivity of TRPM7 to cations, polyamines, and PIP2 interaction and that certain substitutions at this site can disrupt this interaction. Mutations by which PIP2–channel interactions are potentially strengthened  are also more difficult to disrupt by the screening cations. Indeed, we were able to inhibit GOF mutant currents only by raising [Mg2+] to 2.9 mm and higher (see “Results”). It is notable that the positive regulator of TRPM7 channel activity (PIP2) and the negative regulators  share the same site of action, suggesting that Mg2+, polyamines, and pH exert their inhibitory actions through the same mechanism: electrostatic screening (shielding) of the PIP2-negative charge  and threonine (140 Å2) mutants are similar to WT serine (115 Å2), whereas larger side chains  with respective surface areas of 150, 180, 190, 200, and 225 Å2 make the channel insensitive to Mg2+ and other cations (D). Acidic glutamate (190 Å2) and uncharged glutamine (180 Å2) behave similarly, having a similar size. The cutoff appears somewhere between 140 and 150 Å2  because the threonine mutant behaved as the WT, whereas aspartate was a GOF mutant. Surprisingly, the exposure of residue side chains within the truncated TRPM7 model used here changes very little (1% to 4%) despite variations in the size of the mutant side chains at 1107 . For some mutations at position 1107 , energies are extremely large and unfavorable. Others (Asp and Glu) are more modest, but they are also unfavorable and of significant magnitude. This may provide the energetic driving force for a conformational change in the region of residue 1107 that modifies or abolishes PIP2 interactions with the protein. Contributions from electrostatic interactions are not as great as those from nonbonded (steric) interactions and are even stabilizing in the case of Gln and Arg. This suggests that the bulk of the side chains rather than their charge is of greatest consequence. In total, modeling supports functional analyses that indicate that Ala and Cys mutants retain native activity whereas Asp, Glu, Gln, Lys, and Arg do not P2 phospholipid is its main substrate. Several interesting questions about TRPM7 regulation can be addressed in our future experiments. What is the effect of PIP2 depletion at the single-channel level? Mg2+ inhibition consists of a gradual disappearance of conducting channels and a modest, ∼20% reduction in unitary conductance  and C302S (zebrafish) variants were transfected as negative controls. Transient transfection of plasmid DNA was performed with TransIT-LT1 reagent  according to the manufacturer's recommendations. 2–3 days after transfection, the cells were lifted and transferred to glass-bottom recording chambers. In one series of experiments, the transfection mixture was kept in the wells until the start of recordings (∼48 h) , whereas in the other series, it was washed away by changing the culture medium after ∼24 h. We found that, after ∼48 h of uninterrupted transfection, the break-in  and maximum current amplitudes were both markedly reduced in WT- compared with CiVSP C363S–transfected cells, resulting in many cells having no detectable endogenous TRPM7 currents . We therefore conducted our experiments using moderate transfection conditions by removing the transfection reagent after ∼24 h.The HEK293 cell line was maintained in RPMI 1640 medium  supplemented with 10% heat-inactivated fetal bovine serum  and penicillin/streptomycin (HyClone) in a cell culture incubator  at 37 °C and 5% COeviously . Cells wThe murine TRPM7 coding sequence  and its Serine 1107  of murinF). For recording endogenous TRPM7 currents, the internal (pipette) solution contained 106 mm l-glutamic acid, 8 mm NaCl, 5 mm CsF, 10 mm HEDTA, and 10 mm HEPES acid (pH adjusted to 7.3 with CsOH). For the experiments described in m and 150 μm free Mg2+, glutamic acid and HEDTA concentrations were lowered to 101 mm and 8 mm, respectively. For recordings of overexpressed mTRPM7 and its point mutants . In all experiments . 1.0 m MgCl2 and CaCl2 standard solutions  were used for buffer preparation. 20 mm sodium aspartate was replaced with 20 mm sodium propionate in the standard 2 mm Ca2+ buffer to perform the experiments described in Whole-cell patch clamp recordings were performed as described previously , 20. PipFig. 6 mutants  and 8, tm aspartic acid, 6 mm HEDTA, and 10 mm HEPES (pH 7.3 with CsOH) or 140 mm sodium aspartate, 6 mm HEDTA, and 10 mm HEPES (pH 7.3 with NaOH). Deionized water  was used in preparation of recording solutions. Osmolalities were measured with a freezing point depression osmometer  and adjusted by adding D-mannitol. Free Mg2+ concentrations were calculated with Webmaxc software.For Endogenous HEK cell and heterologous TRPM7 channel currents  were evoTRPM7 amplitudes recovered again after initial inhibition by 150 μm Mg2+ (data not shown). This rebound phenomenon was only observed in C363S- but not WT VSP–transfected cells. Such cells were excluded from our analysis.Occasionally, endogenous Im KCl, 50 mm K2SO4, 7 mm MgCl2, 1 mm CaCl2, and 10 HEPES (pH 7.3) to a final concentration of ∼240 μg/ml. The external solution contained 77 mm sodium aspartate, 62.5 mm NaCl, 2 mm CaCl2, 4.5 mm KCl, 10 mm HEPES–sodium, and 0.5 mm glucose (pH 7.3). Membrane potentials were recorded in current clamp mode continuously for ∼1–6 min (10-kHz sampling rate) with Imembrane = 0 after access resistances dropped to 20 MOhm or lower. For recording TRPM7 currents (A), the perforated patch solution contained Cs+ instead of K+. 7 mm Mg2+ present in this solution was sufficient to inhibit TRPM7 currents when whole-cell break-in occurred spontaneously. All experiments were performed at room temperature (∼25 °C).Membrane potentials of untransfected HEK293 cells were measured using the perforated patch technique, essentially as described previously . Brieflycurrents A, the peA partial structural model for mouse TRPM7 was developed using homology modeling methods implemented by Phyre2 , 75. Thel-glutamic acid, HEPES, EGTA, and HEDTA were from Acros Organics . D-mannitol, NaOH, and DMSO were from Fisher Scientific. All other salts and 2-APB were purchased from Sigma-Aldrich. 200 mm stock solution of 2-APB was prepared in DMSO and diluted in the external recording solution on the day of the experiment. Analysis was done by comparing data collected from groups of cells transfected with WT, inactive VSP mutants, and/or mTRPM7 constructs and presented as scatterplots and bar graphs of means ± S.E. Patch clamp data curve fitting and graphing were performed using Origin versions 8, 8.6, and 2016 . Numbers of cells in graphs are given in parentheses. Statistical differences were compared using analysis of variance, Student's t test, and Tukey's multiple comparisons test.T. Z., K. B. W., P. B., and J. A. K. data curation; T. Z., P. B., M. M., and J. A. K. investigation; T. Z., P. B., G. M. A., and J. A. K. methodology; K. B. W., P. B., and J. A. K. formal analysis; G. M. A. software; G. M. A. visualization; M. M. resources; J. A. K. conceptualization; J. A. K. supervision; J. A. K. funding acquisition; J. A. K. validation; J. A. K. writing-original draft; J. A. K. project administration; J. A. K. writing-review and editing.


Prone positioning (PP) during veno-venous ECMO is feasible, but its physiological effects have never been thoroughly evaluated. Our objectives were to describe, through electrical impedance tomography (EIT), the impact of PP on global and regional ventilation, and optimal PEEP level.2O driving pressure and EIT-based “optimal PEEP”. Before, during and after a 16-h PP session, EIT-based distribution and variation of tidal impedance, VTdorsal/VTglobal ratio, end-expiratory lung impedance (EELI) and static compliance were collected. Subgroup analyses were performed in patients who increased their static compliance by ≥ 3 mL/cmH2O after 16 h of PP.A monocentric study conducted on ECMO-supported severe ARDS patients, ventilated in pressure-controlled mode, with 14-cmHn = 21), tidal volume and EELI were redistributed from ventral to dorsal regions during PP. EIT-based optimal PEEP was significantly lower in PP than in supine position. Median (IQR) optimal PEEP decreased from 14 (12–16) to 10 (8–14) cmH2O. Thirteen (62%) patients increased their static compliance by ≥ 3 mL/cmH2O after PP on ECMO. This subgroup had higher body mass index, more frequent viral pneumonia, shorter ECMO duration, and lower baseline VTdorsal/VTglobal ratio than patients with compliance ≤ 3 mL/cmH2O (P < 0.01).For all patients (Although baseline tidal volume distribution on EIT may predict static compliance improvement after PP on ECMO, our results support physiological benefits of PP in all ECMO patients, by modifying lung mechanics and potentially reducing VILI. Further studies, including a randomized–controlled trial, are now warranted to confirm potential PP benefits during ECMO. Despite improvements in the management of severe acute respiratory distress syndrome (ARDS), in-hospital mortality remains high, exceeding 40% . Some paProne positioning (PP) is an effective first-line intervention to treat ARDS , as it iAlthough the use of PP combined with ECMO is still controversial , few stuIn that context, electrical impedance tomography (EIT) could be a promising tool to describe the respective impacts of PP on regional ventilation and possible change of the “optimal PEEP” level on PP. Indeed, EIT is bedside, real-time, non-invasive, functional and radiation-free imaging of the lungs, which provides a regional dynamic lung analysis , 16. ItsWe hypothesized that EIT could help to monitor the impact of PP on regional ventilation and optimal PEEP highlighting potential beneficial effects to prone ECMO-treated severe ARDS patients. The aims of our study on ECMO-supported severe ARDS patients were therefore to describe (1) the PP impact on regional ventilation; (2) the PP influence on the optimal PEEP level; and (3) to identify different EIT patterns depending on static compliance gain.We conducted this study during a 4-month period in our 26-bed medical intensive care unit (ICU). Written informed consent was obtained from all patients’ surrogates before inclusion. The study was approved by the appropriate legal and ethical authorities .Inclusion criteria were: ARDS patient on VV-ECMO, pressure-controlled mechanical ventilation mode and sedated with a Richmond Agitation–Sedation Scale ≤ − 2. Exclusion criteria mainly reflected clinical contraindications to using EIT, PP or high PEEP levels , and after 16 h of PP. To reach a steady-state, we first performed a recruitment maneuver, applying 40 cmHfor 40 s . Then, aescribed , 20. Thuescribed , was ideescribed .2O) and a constant respiratory rate of 20/min. PP lasted 16 h. EIT data were recorded before PP, immediately after PP, after 1, 6, 14 and 16 h of PP, respectively, after returning to SP, and after 6 h in SP. A recruitment maneuver, consisting of sustained inflation at 40 cmH2O for 40 s [Briefly, patients were ventilated at previously estimated optimal PEEP with constant driving pressure  [2O . Lastly, fluid balance throughout the protocol was noted.At each step, hemodynamic parameters, VT, corrected minute ventilation (minute ventilation × PaCO2/40) , arteriadorsal/VTglobal  [EIT analysis is detailed in the Supplemental Digital Content. Briefly, lung images were divided into two symmetrical non-overlapping ventral and dorsal horizontal regions of interest (ROIs) . The verregions) –25, and regions) ). Notablregions) , EELI caregions) , 28  after 16 h of PP, defined by an increase ≥ 3 mL/cmH2O compared to baseline  [2O with driving pressure at 14 cmH2O would result in a VT gain of ≥ 42 mL.Because it would have been difficult to discern the direct PP influences on gas exchanges under ECMO, we arbitrarily decided not to use PaO.e., SP) , 10, 29,P < 0.05 required to assert the presence of a difference.Statistical analyses were computed with Prism 4.01 software . Because all parameters had non-normal distributions, data are expressed as median . Friedman ANOVA for repeated measures was used to compare data obtained at each step, followed, when appropriate, by pairwise comparisons using a Dunn post hoc test with Bonferroni correction. Qualitative data were compared with Fisher’s exact test. All comparisons were two-tailed, with Twenty-one patients (median (IQR) age 56 46–61) years, 62% male) were included; their main characteristics are reported in Table  years, 62O (P < 0.01), corresponding to a VT and a corrected minute ventilation increase from 4.2 (3.3–5.4) to 5.6 (3.8–6.4) mL/kg and 5.5 (4.3–6.8) to 6.2 (5.1–6.9) L/min (P = 0.04), respectively. Consequently, local compliance variations, which is calculated based on local impedance variation compared to baseline, increased by 120 (60–180) % at the end of PP . Their global static compliance increased from 25 (19–31) to 35 (27–39) mL/cmH2O, resulting in an increase of the VT and the corrected minute ventilation from 5.1 (3.4–5.9) to 6.3 (5.6–6.7) mL/kg, and from 6.2 (4.8–6.8) to 6.6 (5.3–7.4) L/min, respectively. These benefits remained significant 6 h after return in SP (P ˂ 0.0001)  mmHg to 31 (29–37) mmHg after a PP session (p = 0.03) without any variation of sweep gas flow  to 27 (20–37) mL/cmHP < 0.05), with VTdorsal/VTglobal ratio significantly higher after the 16-h PP session versus baseline  and 69 (36–76)% (P < 0.05) . It was significantly reduced after PP in all patients, shifting from 14.8 (12–17) to 11.7 (9–14) cmH2O for PCG+ , while PCG−, optimal PEEP declined from 12 (8–15.5) to 9 (6.5–11.5) cmH2O  EIT-estimated optimal PEEP decreased with PP, highlighting the potential reduction of atelectasis with PP.To our knowledge, PP physiological effects in a population of patients with severe ARDS on ECMO have not yet been thoroughly studied. Based on EIT-monitoring, the main findings were: (1) although PCG+ and PCG− had different tidal volume distribution before PP, all patients with severe ARDS on ECMO exhibited a shift of the VT distribution and EELI from the ventral-to-dorsal ROIs resulting in an increased of the local compliance and the VT2 improvement, other ventilatory monitoring tools, such as thoracic tomodensitometry) [2, which is, a well-known marker of PP response [By defining PP “responders” on PaOtometry)  or lung tometry) , have faresponse , 30. In These data suggest that EIT could offer a promising bedside, dynamic, non-invasive, functional analysis of lung mechanics that could predict and monitor potential PP “response” on ECMO. Our results mainly underscored immediate PP effects, which continued to evolve during the 16-h procedure, illustrating the need for long PP sessions to obtain the best benefits , 32. Con2-based beneficial PP effects mainly depend on the lung recruitment/derecruitment ratio [Baseline lung mechanics and predominant lesions differed between PCG+ and PCG− patients, and could explain these different responses between subgroups. Indeed, baseline tidal impedance were mostly distributed in ventral ROIs only in PCG+ . This finding suggests a collapse of the dorsal lung ROIs and functional aerated ventral ROIs in PCG+ , whereas ventral ROIs were over distended in PCG− at baseline. Consistently, previous observations reported that PaCOnt ratio , 33. In Based on these results, should we decide to perform PP based on predicted PP-response for ECMO patients? The relevance of this question appears low. Indeed, the increase of EELI in the dorsal regions was observed in all patients, as previously reported with other tools , 35 and One of our main results is the impact of PP on optimal PEEP evaluated with EIT. EIT-based optimal PEEPs decreased significantly after 16 h of PP, highlighting the remarkable PP impact on respiratory mechanics. To date, this aspect of PP management has only been studied in patients with healthy lungs and yielded controversial results. Spaeth et al.  reportedOur study has several limitations. First, EIT provides only a cross-sectional lung-region evaluation, which may differ from whole lungs . This diEIT monitoring of PP during VV-ECMO shows that PP impacts global and regional ventilation, illustrated by a progressive redistribution of VT and EELI from ventral to dorsal regions. Although baseline VT distribution on EIT may predict static compliance improvement after PP on ECMO, our results support physiological benefits of PP in all ECMO patients, by modifying lung mechanics and potentially reducing VILI. In addition, clinicians should be also encouraged to reevaluate PEEP level during PP to avoid overdistension. Further studies, including a randomized–controlled trial, are now warranted to confirm potential PP benefits during ECMO.Additional file 1. Details about exclusion criteria, EIT data acquisition and analysis, and exclusion of the out-of-phase regions.


Acute renal ischemia/reperfusion (I/R) injury is a clinical condition that is challenging to treat. Meldonium is an anti-ischemic agent that shifts energy production from fatty acid oxidation to less oxygen-consuming glycolysis. Thus, in this study we investigated the effects of a four-week meldonium pre-treatment (300 mg/kg b.m./day) on acute renal I/R in male rats (Wistar strain). Our results showed that meldonium decreased animal body mass gain, food and water intake, and carnitine, glucose, and lactic acid kidney content. In kidneys of animals subjected to I/R, meldonium increased phosphorylation of mitogen-activated protein kinase p38 and protein kinase B, and increased the expression of nuclear factor erythroid 2-related factor 2 and haeme oxygenase 1, causing manganese superoxide dismutase expression and activity to increase, as well as lipid peroxidation, cooper-zinc superoxide dismutase, glutathione peroxidase, and glutathione reductase activities to decrease. By decreasing the kidney Bax/Bcl2 expression ratio and kidney and serum high mobility group box 1 protein content, meldonium reduced apoptotic and necrotic events in I/R, as confirmed by kidney histology. Meldonium increased adrenal noradrenaline content and serum, adrenal, hepatic, and renal ascorbic/dehydroascorbic acid ratio, which caused complex changes in renal lipidomics. Taken together, our results have confirmed that meldonium pre-treatment protects against I/R-induced oxidative stress and apoptosis/necrosis. Acute renal ischemia/reperfusion (I/R) is a temporary restriction of kidney blood supply, followed by blood flow restoration and re-oxygenation. As the main cause of acute kidney injury, it may occur due to aortic dissection, surgery, infarction, sepsis, or organ transplantation . Due to Meldonium is an anti-ischemic drug clinically used to treat myocardial and cerebral ischemia . MeldoniTo address this issue, the aim of the present experiment was to investigate the effects of four-week meldonium pre-treatment with 300 mg/kg b.m./day of rats subjected to a well-established experimental model of renal I/R, with ischemia lasting for 45 minutes, followed by 4 hours of reperfusion . The chaThere is little literature on the effects of meldonium on body mass gain and food and water intake, except for two studies. Porter et al.  showed tOur measurement of body mass and food and water intake were completed before the surgical procedures (sham or IR), meaning that all detected changes were caused by meldonium and not by surgical procedures. As can be seen from our results, meldonium decreased body mass gain B,E and fThe evidence that meldonium actually shifts metabolism towards a better utilized glucose are that there are changes in the kidney content of carnitine, glucose, and lactic acid . While IDuring I/R, depleted oxygen supply causes dysfunctionality of the ATPase-dependent ion transport and intracellular ATP. Further, pH levels decrease, intracellular and mitochondrial calcium levels increase, and there is a disturbance in numerous signaling pathways . Upon reTo examine the effect of meldonium pre-treatment on the extent of apoptosis activation in the kidney subjected to I/R, we measured the changes in the Bax/Bcl2 ratio. Our results showed that I/R increased the Bax/Bcl2 ratio by 2.7-fold and that meldonium reduced this increase for 35% A. This fApoptotic and necrotic cell death occur simultaneously under I/R conditions. Cellular death due to necrosis result in the loss of cell membrane integrity and uncontrolled release of damage-associated molecular pattern molecules (DAMPs) into the extracellular space. HMGB1, which belongs to the DAMPs family, diffuses out of stressed, damaged, or dying cells, which is why it serves as necrotic marker . Our resIt is known that I/R elevates ROS generation and decreases antioxidant defense through antioxidative enzyme gene downregulation. Studies have reported the loss of the MnSOD enzyme activity following I/R, with the MnSOD inactivation prior to the onset of renal damage, which suggests an upstream of oxidative damage to renal damage . It has We also investigated the protein expression of Nrf2, which has been shown to be the most important inducible transcription factor that exerts protective effects against renal ischemic damage by regulating the endogenous antioxidant system. Once activated in cytosol, Nrf2 trans-activates antioxidant response elements, which further promote the CuZnSOD, GSH-Px, GST, CAT, and HO-1 expression increase . SeveralUnexpectedly, changes in Nrf2 expression were not accompanied by appropriate changes in the activity of Nrf-2 associated antioxidant enzymes. As can be seen from We also investigated the ratio between ascorbic acid (the reduced form of vitamin C) and its oxidized form (dehydroascorbic acid). The AA redox reactions begin with a single-electron oxidation that generates a mono-dehydroascorbate radical (MDHA), which further dismutates DHA plus AA . Both MDAs can be seen in However, it still remains uncertain how renal I/R, which we causes kidney oxidative stress , may cauIt is an interesting finding that I/R and meldonium only impact NA content in adrenal glands without changes in adrenaline content . While IRenal necrosis occurs secondary to ischemia . CellulaLipids are fundamental components of biologic membranes. Depending on the chemical structure and biosynthesis, lipids can be divided into eight categories: fatty acids (FA), glycerolipids, sphingolipids, phospholipids, saccharolipids, sterol lipids, prenol lipids, and polyketides . One of cis and trans Δ9-octadecenoic, C18:1 c + t), linoleic/linolelaidic , behenic , cervonic , and euric . Under applied chromatographic conditions, cervonic and euric acids coelute at the same retention time, so their concentrations were calculated as the sum. The same was the case for cis- and trans- forms of C18:1 and C18:2, so the results were presented as the 10 fatty acids analysis , pentadecylic , palmitic , palmitoleic , margaric , stearic , oleic/elaidic , with a decrease only in the case of C14:0 and C16:0, and no changes in the case of C18:0. Interestingly, changes caused by meldonium depend both on treatment and/or FA type. For example, in sham operated animals, meldonium generally increased concentration of almost all tested fatty acids, with the exception of C22:0 and C22:6 + C22:1, whose concentrations were not changed. However, in animals subjected to I/R, meldonium in some cases caused changes that corresponded to those in the S + M group, such as the concentration increase of C14:0, C16:0, C18:0, C18:1 c + t, and C18:2 c + t. This was not the case with C15:0, C16:1, and C17:0, whose concentrations meldonium decreased in the I/R + M group, which was in contrast to the increase it caused in S + M group. Similarly, in I/R animals meldonium caused a C22:0 and C22:6 + C22:1 concentration decrease, which was in contrast to sham operated animals where no chances were caused. To explore potential similarities and differences between fatty acid profiles across all experimental groups the principal component analysis was applied . The datAs can be seen from our results, the sum of C22:6 and C22:1 exhibits the highest positive value, whereas C16:0 exhibits the highest negative value B. The grThe small- and medium-chain-fatty acids are supposed to be able to enter the mitochondria by diffusion, whereas long chain fatty acids (C14–C20) require a transport system . In mitoAll animal procedures were performed in compliance with the ARRIVE guidelines and Directive 2010/63/EU. In accordance with the National legislation, all animal procedures were approved by the Veterinary Directorate of the Ministry of Agriculture and Forestry and Water Management, License number 323-07-07137/2018-05 (30.07.2019).Rattus norvegicus) male rats weighing 341.63 ± 4.95 g were used for the experiment. The animals were acclimated to 22 ± 1 °C and maintained under a 12 h light/dark period. The rats were randomly divided into four groups  and housed two per cage for four weeks on ad libitum access to a standard diet  and tap water (with or without meldonium). In total, we used 32 rats. Rat groups were as follows: animals that drank tap water for four weeks and then sham operated (S group); animals that drank tap water with meldonium for four weeks and then sham operated (S + M group); animals that drank tap water for four weeks and then I/R operated (I/R group); and animals that drank tap water with meldonium for four weeks and then I/R operated (I/R + M group).Wistar strain  propionate; THP; MET-88)  was dissolved in tap water in concentrations ranging from 2–3 mg/ml. Depending on the water intake, meldonium concentrations in the water were adjusted weekly to achieve its consumption of around 300 mg/kg b.m./day. Based on the four-week measurement, the meldonium consumption was 302.01 ± 4.44 mg/kg b.m./day in sham operated animals and 301.42 ± 3.67 mg/kg b.m./day in I/R operated animals.Body mass, body mass gain, and food and water intake were measured weekly throughout the experiment. The results were expressed as a time course of measured values and recalculated into the area under a curve (AUC) values.Anesthetized rats were subjected to a non-recovery surgical procedure described elsewhere . In brieIschemia was produced using a reversible clamp that held both renal pedicles with the appropriate arterial clip for 45 min. At the beginning of reperfusion, clips were removed with big forceps and kidneys were subjected to reperfusion lasting for four hours. At the end of four-hour reperfusion, rats were euthanized using an overdose of thiopental sodium.Blood and tissue samples were collected for further analysis immediately after euthanasia.× g for 10 min in a refrigerated centrifuge. The resulting supernatant was immediately transferred into a clean polypropylene tube using a Pasteur pipette [Blood was taken into the heparin-coated Eppendorf tubes directly from the intravenous catheter and inserted into the jugular vein, then incubated at room temperature for 45 min to allow clot formation. Clots were removed via centrifugation at 2000 pipette .The liver, kidneys, and the adrenals of the rats were isolated and dissected out within 3 min, then placed in ice-cold 155 mmol NaCl and washed with the same solution. One part of the kidney was placed in formaldehyde for further histological analysis. The serum and the tissue samples for the rest of the analysis were stored at −80 °C until the analysis.Tissue sample (50 mg) was mixed with 2 mL of ultra-pure water and pulverized with a tissue grinder. After extraction in an ultrasound bath at 0 °C, samples were centrifuged at 12,000 rpm. The supernatant was transferred in 10 mL normal flask and diluted with ultra-pure water to the mark. The sample solutions were kept at −80 °C till analysis.® Chromatography Workstation with Chromeleon 6.7.2 Chromatography Management software. The separation was performed on IonPac AS15 Analytical, 4 × 250 mm (P/N 053940) and IonPac AG15 Guard, 4 × 50 mm (P/N 053942) column. The mobile phase flow rate was set to 1000 ml/min, while the concentration of potassium hydroxide was changeable to achieve the following gradients: 0–15 min 10 mM KOH; 15–25 min 10–45 mM KOH; 25–26 min 45 mM KOH; 26–31 min 45–10 mM KOH; and 31–36 min 10 mM KOH. The column temperature was termostated at 30 °C. The conductivity cell temperature was 35 °C. The suppressor current was 134 mA. The backpressure was ~2200 psi.The standard of lactic acid was purchased from Sigma Aldrich. Ion chromatography was used to assay the appearance and quantification of lactic acid. For this purpose, a Dionex ICS-3000 chromatographic setup was used, which consisted of a single pump, a conductivity detector (ASRS ULTRA II (4 mm) (P/N 061561), recycle mode), a eluent generator (potassium hydroxide (KOH) (P/N 058900)), and a Chromeleon®PA100 pellicular anion-exchange column (4 × 250 mm)  at 30 °C. The mobile phase consisted of the following linear gradients : 0–5 min, 15% A, 85% C; 5.0–5.1 min, 15% A, 2% B 83% C; 5.1–12 min, 15% A 2% B 83% C; 12–12.1 min, 15% A, 4% B, 81% C; 12.1–20 min 15% A, 4% B, 81% C; 20–20.1 min 20% A, 20% B 60% C; 20.1–30 min 20% A, 20% B 60% C; where A was 600 mM sodium hydroxide, B was 600 mM sodium acetate, and C was ultrapure water. Previously, the analysis system was preconditioned at 15% A and 85% C for 15 min. Each sample (25 µL) was injected with an ICS AS-DV 50 auto sampler . The electrochemical detector consisted of gold as working and Ag/AgCl as reference electrode.The standard glucose was purchased from Tokyo Chemical Industry, TCI, . Sodium hydroxide and sodium acetate trihydrate were obtained from Merck . All aqueous solutions were prepared using Ultrapure TKA deionized water. The standard solution of glucose was prepared in ultrapure water at 100 ng/mL concentration. Calibration standards were prepared from stock solution by dilution with ultrapure water. The quality control mixture used for monitoring instrument performance was prepared by diluting concentrations of 20 ng/mL. Chromatographic separations were performed using DIONEX ICS 3000 DP liquid chromatography system  equipped with a quaternary gradient pump  and a Carbo Pacv/v). A classical chromatographic chamber  was filled with 3 mL of mobile phase made by components of analytical grade of purity. The chamber was saturated with solvent for 30 min. All experiments were performed on ambient temperature. The plates were applied with CAMAG Linomat 5 with 2 µL aliquots of previous prepared and defrosted aqueous solutions. The plates were scanned using CAMAG TLC Scanner 3 at 260 nm. Chromatograms were analyzed using winCATS software version 1.4.2.8121.L-Carnitine was obtained from Sigma Aldrich. L-Carnitine (10 mg) was weighted in 10 mL of a normal flask diluted with ultra-pure water. Next, an ascending thin-layer chromatography was performed as an adsorbent on RP-18 silica . The chromatograms were developed using an acetonitrile-water binary mixture with a ratio of 1/1 . The carrier gas used in this experiment was helium (1 mL/min) and the injected volume of the sample was 1 µL. The initial temperature for the oven was 50 °C (1 min), then 25 °C/min to 200 °C, and immediately put to 3 °C/min to 230 °C (held for 18 min). The injector was in split mode (50:1) while the temperatures of injector, transfer line, and ion source were 250, 260, and 260 °C, respectively.g for 90 min and sonicated for 30 s at 10 kHz on ice (Bandeline Sonopuls HD 2070), followed by centrifugation in a Beckman ultracentrifuge at 100,000× g for 90 min at 4 °C. The obtained supernatants were used for biochemical analyses.The kidney samples were minced and homogenized in 10 volumes of 25 mmol/L sucrose. They contained 10 mmol/L Tris-HCl, had a pH of 7.5 at 1500 rpm, and used a Janke and Kunkel  IKA-Werk Ultra-Turrax homogenizer at 4 °C. Homogenates were centrifuged at 4 °C at 100,000× 2O2/min/g wet mass. The activity of GSH-Px was measured following the oxidation of nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate at 340 nm with t-butyl hydroperoxide [The activity of antioxidant defense enzymes was measured simultaneously in triplicate for each sample using a Shimadzu UV-1800 spectrophotometer and a temperature-controlled cuvette holder. The total SOD activity was measured in the supernatant by using the adrenaline method, which is based on the SOD capacity to inhibit adrenaline autoxidation to adrenochrome . To deteperoxide  and exprperoxide  and exprperoxide  and exprperoxide , which wperoxide  and exprperoxide , which hThe serum, liver, and adrenal concentration of AA and DHA were measured according to the method established by Nováková et al. . L-AscorA stock standard solution of L-Ascorbic acid (1 mg/mL) was prepared in 10% MPA and kept at −20 °C. Standard solutions were prepared by diluting the stock standard solution in 10% MPA.The phosphate buffer  was used as the mobile phase. Sodium phosphate monobasic dihydrate (26 g) was accurately weighed, dissolved in approximately 800 mL of water, and the pH was adjusted to a value of 3.0 with a phosphoric acid solution (49–51%). The resulting solution was made up to 1 l with water.v:v). All samples were centrifuged . Supernatants were used for the analysis of native AA concentration. For the analysis of TVC concentration, 100µL of the above supernatant was treated with 300 µL DTT solution (2.5 mg/mL in phosphate buffer) for 30 min at 4 °C for complete conversion of DHA to AA. The mixture was then re-acidified with 200 µL of 10% MPA and transferred to the autosampler unit.Tissue samples were homogenized in 10% MPA (1 mg:10 µL), using an Ultra-Turrax homogenizer, and sonicated (3 × 10 s). Plasma samples were added to a 10% MPA  was pumped over 10 min in an isocratic flow of 800 µL/min. The applied potential for electrochemical measurements was 600 mV and the separation temperature was 25 °C. The 20 µL of samples and standard solutions were applied to the system. The concentration of AA and DHA were expressed as µg/mg tissue and µg/mL serum.2 × 5H2O) were purchased from Sigma-Aldrich. Ammonium formate was supplied by Fisher Scientific, formic acid (49–51%) by Fluka, and methanol by J.T. Baker. Purified water was obtained via a BlueClearRO600P reverse osmosis water cleaner system with an integrated BlueSoft07-MB mixed bed salt remover .The concentration of adrenaline (AD) and noradrenaline (NA) in the adrenal glands was performed according to the method of Stefanovic et al. . Ethylen4; 0.2% MgCl2).Stock standard solutions of catecholamines (1 mg/mL) were prepared in methanol and kept at –20 °C. Standard solutions were prepared by diluting the stock standard solution in DEPROT  was used as one of the mobile phase components. Ammonium formate (6.3 g) was accurately weighed and dissolved in approximately 800 ml of water. Then, the pH was adjusted to the value of 3.6 with formic acid. The resulting solution was made up to 1 l with water.Tissue samples were homogenized in DEPROT (1 mg:10 µL) using an Ultra-Turrax homogenizer. They were then sonicated (3 × 10 s) and centrifuged . Supernatants were transferred in separate tubes and placed in the HPLC system’s autosampler.We obtained the data using a Thermo Scientific (Dionex UltiMate 3000) HPLC system that consisted of a degasser unit, binary pump, autosampler, column compartment, and RS electrochemical detector equipped with a glassy carbon working electrode. An Acclaim Polar Advantage II, C18 (3 µm) HPLC column  was used. Instrument control and data acquisition were carried out using a Chromeleon7 Chromatography Data System (ThermoScientific).The following chromatographic conditions were obtained after the method optimization. The mobile phase that had an ammonium formate buffer  as an A solution and methanol as a B solution was pumped at a flow rate of 500 µL/min with the following gradient. The run was started with a mobile phase consisting of 98% A and 2% B solution. Starting from the ninth minute of the run, the B solution rose to reach 80% in the 13th min. Starting from the 18th min until the end of the run (25th min), the column was re-equilibrated with the initial mixture of each mobile phase solution (2% of A and 98% of B solution). The potential for electrochemical measurements was 850 mV; the separation temperature was 25 °C. Next, 40 µL worth of samples and standard solutions were applied into the system. The concentration of catecholamine was expressed as µg/mg tissue.g in a Beckmann rotor Ti 50  for 90 min. The resulting supernatant was aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C.To prepare for the kidney homogenate, tissue was homogenized in an ice-cold homogenization buffer , which was supplemented with 1× phosphatase inhibitor Mix I and 1× protease inhibitor Mix G (Serva). After sonication, the homogenate was centrifuged at 100 000× Protein samples of serum (1 µL) or kidney homogenates (50 µg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes . These samples were then blocked in a solution  containing 5% non-fat condensed milk. After the protein transfer, PVDF membranes were incubated with a primary antibody for 1.5 h at room temperature. Western immunoblot analysis was performed using Abcam rabbit polyclonal HMGB1 (ab 18256), Nrf2 (ab 92946), HO-1 (ab 13243), β actin (ab 8227), Bax (2772S), Bcl-2 (2876S) antibodies, Santa Cruz Biotechnology rabbit polyclonal MnSOD/SOD-2 (sc 30080), p38 (sc 535), p-p38 (sc 7975-R), Akt 1/2/3 (H-136), pAkt1/2/3 (ser 473)-R antibodies, and a goat polyclonal anti-HMGB1 antibody for serum analysis (K12). The blots were probed with horseradish peroxidase-conjugated secondary Santa Cruz Biotechnology bovine anti-rabbit IgG pr (sc 2379), bovine anti-goat IgG (sc 2378), and Abcam goat anti-rabbit IgG (ab 97051) antibodies. Immunoreactive bands were identified using an enhanced chemiluminescence (ECL) detection system  according to the manufacturer′s instructions. The blots were scanned and the intensities of the signals were quantified using TotalLab  electrophoresis software (ver.1.10). Actin was used as a loading control for the quantification. For re-probing, membranes were incubated in 2% SDS, 100 mM β-mercaptoethanol, and 62.5 mM Tris-HCl pH 6.8 for 35 min at 50 °C, then rinsed three times, blocked, and probed again with another antibody. All immunoblot analyses were obtained from at least three independent experiments.Kidney samples were collected and fixated in 4% formaldehyde solution. After fixation, the samples were dehydrated in a series of increasing ethanol solutions  followed by an immersion in a clearing agent (xylene). The tissue samples were embedded into paraffin wax and cut into 5 µm thick sections. A minimum of 10 fields for each kidney slide were examined using a Leica DM LS 2 type 11020518016 microscope . The histological analysis evaluated tubular necrosis, interstitial oedema, loss of brush border, and casts formation.p < 0.05.Where appropriate, the results were expressed graphically as time-course curves, which were subsequently recalculated into area under a curve (AUC) values. The single time-point measurements were presented as mean ± standard errors of mean. AUC values were presented as the percentage of controls. Data were checked for normality using Lilliefors and Kolmogorov–Smirnov tests. Differences in investigated parameters between groups were calculated using one-way ANOVA. When significant differences were found, pairwise comparisons were performed using Holm-Sidak post hoc tests. The statistical package SIGMAPLOT was used for all the analyses and graphical presentations, except for the principal component analysis (PCA), which was performed using the NCSS 2004 software package. The level of statistical significance was defined as