The ratio of HIF1A to actin alerts was calculated utilizing the Western Potassium clavulanateblotting benefits (Fig 1A). Be aware the higher expression of HIF1A in EBV-positive cells.Statistical evaluation of relative HF1A protein expression. Kruskalallis assessments ended up applied to four groups, comprising 7 EBV-damaging, six Latency I, seven Latency III BL mobile lines, and four LCLs. HIF1A was expressed considerably greater in Latency III cells (p = .008<0.05).Expression levels of genes involved in the glycolytic pathway. We studied isogenic BL41 (Bl41, EBV negative BL41/B95.8, Latency III) and Mutu (Mutu, EBV negative Mutu I, Latency I and Mutu III, Latency III) BL cell lines, and LCL121028 cells (5 months old). No significant differences in expression levels were observed in the five groups of cells for the studied genes. Each point represents the median value for three Q-PCR reactions. No value differed by more than 30% of the means.GLUTI, LDHA, MCT4, PDK1, PGK1, and PKM2 genes were moderately high and slightly higher in Latency III cells, in comparison with Latency I and EBV-negative cells. However, this difference was not significant in Kruskalallis tests (p = 0.1210>.05). Every single point on Fig 3 represents a median value for 3 Q-PCR reactions. Notice that MCT4 expression was quite higher in BL mobile traces. This implies the enhanced production of lactate and its export from the cells.The concentrations of lactate and pyruvate ended up measured by spectrophotometry (Desk one). The concentration of pyruvate was increased in BL strains than in LCLs (Fig 4). This implies that BLs may possibly proliferate quicker than LCLs. All studied mobile lines made equally high ranges of lactate. There have been no dramatic variations in the lactate dehydrogenase (LDHA) expression levels at the mRNA stage (Fig four). Nevertheless, the LDHA sign was slightly increased in Latency III BL cells and LCLs, and the catalytic activity of LDHA enhanced considerably in the Latency III biochemical characteristics of BL cell traces. The BL cells described in Fig three and LCL121028 cells ended up assayed for the concentrations of l-lactate and pyruvate, and for lactate dehydrogenase (LDHA) catalytic activity by colorimetric assays (see Supplies and techniques area). Latency III BL cells showed drastically greater amounts of pyruvate production and LDHA activity (p = .0091 and p = .0349, respectively). There have been no variances in LDHA expression at mRNA amounts (no value differed more than 30% from the means). Lactate concentrations were related in all five studied groups.BL mobile strains (Fig 4). This could be explained by partial HIF1A protein stabilization effected by the EBNA-3 and EBNA-five expressed by these cells.To check whether HIF1A may well be dependable for the transcriptional activation of genes involved in regulation of aerobic glycolysis in BL cells of Latency III, the transactivating capability of HIF1A was inhibited. It is known that this transcription factor is active only as a heterodimer with ARNT [18]. Hence, formation of a protein intricate amongst HIF1A and ARNT was prevented, utilizing acriflavine hydrochloride (ACF) to inhibit this binding [19]. To examination mobile viability, cells had been developed in medium that contains five M ACF. Cells were collected at 18 hours, and the numbers of residing cells had been counted. Enormous cell demise was noticed right after twenty h (Fig 5). To avoid any affect of mobile dying on Q-PCR outcomes, probes ended up gathered soon after 3 h of remedy with ACF. All the analyzed HIF1A-dependent genes (GLUTI, HK, LDHA, MCT4, PDK1, PGK1, and PKM2) were drastically downregulated in LCLs right after therapy with ACF (p<0.05, Fig 6). This suggests that ACF could inhibit the transactivating ability of HIF1A. However, there were no statistically significant changes in the expression levels of the same genes in different BL cell lines (Fig 6).Influence of ACF on the proliferation of BL cell lines and LCLs. The percentage of living cells is presented as a function of the time of treatment. LCL121028 cells were quickly killed by ACF.It was reported earlier [20] that 2-methoxyestradiol (2-MeOE2) inhibited nuclear translocation of HIF1A and HIF1B, and, as consequence, transcription of the HIF1A-dependent genes decreased. Expression of a set of genes (GLUTI, HK, LDHA, PDK1, PGK1, and PKM2) was assessed in BL cell lines and LCL121028 upon the treatment with 5 M of 2-MeOE2 (see S2 Table). And again, expression of the abovementioned genes was decreased significantly (p = 0.0001) only in LCLs, but not in BL cell lines. Thus, HIF1A played a limited role in the transactivation of genes involved in aerobic glycolysis in BL cell lines, in contrast to LCLs.As mentioned above, BLs are characterized by an upregulated expression of MYC, arising from chromosomal rearrangements [21]. In addition, MYC induces a set of genes involved in glycolysis [11]. Hence, our next question was whether inhibition of the transactivating ability of MYC would result in the downregulation of those genes. The agent 10058-F4 (5-[(4-ethylphenyl)methylene]-2-thioxo-4-thiazolidinone) was used to abolish the transactivating ability of MYC. This prevents dimerization of the MYC and MAX proteins, thus inhibiting the transactivation of MYC-dependent genes [22]. Cell growth medium was supplemented with 100 M of 10058-F4. Cells were treated for 4 h, and the expression levels of genes involved in glycolysis (GLUTI, HK, LDHA, MCT4, PDK1, PGK1, and PKM2) were assessed by Q-PCR. Genes responsible for the Warburg effect were downregulated in all the BLs regardless of the presence or absence of EBV and the type of latency (Fig 7). Importantly, no significant decreases in gene expression levels were observed in LCLs in the same conditions (Fig 7). Similar results were obtained when 10074-G5, another MYC-inhibitor (described in [23]) was used (S2 Table). This suggests that MYC functions as a transcription factor to activate genes involved in aerobic glycolysis in BL cells but not in LCLs. In other words, HIF1A is the main regulator of the Warburg effect in LCLs, consistent with our previous data [10].Expression levels of genes involved in glycolysis upon treatment with ACF. Expression levels of a set of genes (GLUTI, HK, LDHA, MCT4, PDK1, PGK1, and PKM2) were assessed by Q-PCR after the treatment of cells with 5 M of ACF for 3 h. Kruskalallis tests were applied to the results for 12 groups namely, two EBV-negative, one Latency I, two Latency III BL cell lines, and LCL121028 cells--controls and treated with ACF. Note the significant decrease in gene expression under ACF treatment in LCLs (p = 0.0082 framed in the Fig). The median value for three Q-PCR reactions is shown the standard deviation did not exceed 30% of the means.Next, the LDHA activity was assayed in a set of BL cell lines and LCLs, under normal conditions and with 10058-F4 treatment. Four groups of cells were studied namely, EBV-negative BL cell lines (Ramos, Bl28, DG75), Latency I (BL28/B95.8, Rael, Akata (+)), and Latency III (BL16, BL18, RAJI), and three LCLs (051128, 111210, and 120214). The LDHA activity was decreased in all the BLs upon MYC inhibition but not in the LCLs (Fig 8). This suggests that aerobic glycolysis is controlled mainly by MYC in BL cell lines, in contrast to HIF1A in LCLs.As mentioned above, the Warburg effect is one of the hallmarks of cancer cells that can also be observed in rapidly proliferating cells such as activated T-cells [24]. We have shown previously that LCLs can also exhibit the Warburg effect thus, lymphoblastoid cells show features that are characteristic for malignant and also for rapidly proliferating cells [10]. We have also shown that stabilized HIF1A transactivated those genes involved in glycolysis. Stabilization of the HIF1A protein was caused by the lack of hydroxylation from inactivation of prolylhydroxylases PHD1 and PHD2. The catalytic activity of PHD1 and PHD2 is inhibited because they form protein complexes with the EBV-encoded nuclear antigens EBNA-5 and EBNA-3, respectively. The molecular regulation of aerobic glycolysis in BL cells is not yet fully understood. Using the model BL cell line P493-6 that carries an inducible MYC repression system, it was suggested that at least two genes encoding enzymes for glycolysis (HK2 and PDK1) are regulated by the HIF1A and MYC proteins cooperatively [25]. To elucidate glycolytic pathway regulation in BLs, we studied the expression patterns of GLUTI, HK, LDHA, MCT4, PDK1, PGK1, and PKM2 encoding enzymes for glycolytic pathway. All these genes were expressed at high levels in BL lines and LCLs (Fig 3). Therefore, what is the main regulator of expression in BL lines expression levels of genes involved in glycolysis upon treatment with 10058-F4. Expression levels of a set of genes (GLUTI, HK, LDHA, MCT4, PDK1, PGK1, and PKM2) were assessed by Q-PCR after the treatment of cells with 100 M of 10058-F4 for 4 h. Kruskalallis tests were applied to the results for 12 groups: two EBV-negative, one Latency I, and two Latency III BL cell lines, and LCL121028 cells--controls and treated with 10058-F4. Note the significant decreases in gene expression levels under 10058-F4 treatment in the BL cell lines (p = 0.011), in contrast with no change in LCL121028 cells (encircled). The median value for three Q-PCR reactions is shown the standard deviation did not exceed 30% of the means.We found that the HIF1A protein was expressed at high levels in EBV-positive BLs, especially in Latency III cells (Figs 1 and 2). To monitor the importance of HIF1A function in BL cells, the transcriptional activity of HIF1A was inhibited by ACF to retain HIF1A in the cytoplasm, thus preventing HIF1ARNT heterodimer formation [19]. HIF1A-induced genes, such as GLUT1, HK, PGK1, PDK1, MCT4, PKM2, and LDHA were downregulated significantly in LCLs, in contrast to BL cells that showed no such inhibition (Fig 6). It is known that treatment with ACF might lead to the inhibition of lipopolysaccharide-induced NF-B activation [26]. Notably, ACF can induce apoptosis and necrosis in yeast cells [27] therefore, cells were treated with ACF for only 3 h when massive cell death had not yet occurred (Fig 5). The results suggest that HIF1A is not a major factor in the regulation of aerobic glycolysis in BLs. As discussed above, MYC is another transcription factor activating the transcription of genes involved in glycolysis. However, its expression is quite low in LCLs, even though the EBNA-2 protein activates the MYC promoter through interactions with the CBP (cAMPresponse element (CREB)-binding protein, NP_001073315) and histone acetyl transferase P/ CAF (p300/CBP-associated factor NP_003875) [28]. In BL cells, MYC is expressed at high levels because of gene activation by chromosome translocation. To monitor the consequences of inhibition of MYC transactivating ability, LCLs and BLs were treated with 10058-F4 to prevent the binding of MYC and Max proteins. No significant changes in responsive gene expression lactate dehydrogenase activity in BL cell lines and LCLs. LDHA catalytic activity was measured in the control cells and after treatment with 100 M of 10058-F4 for 4 h. The median value of three measurements (standard deviation not exceeding 30%) is shown on the Fig. Each of the four groups consisted of three cell lines: EBV-negative (Ramos, Bl28, DG75), Latency I (BL28/B95.8, Rael, Akata (+)), and Latency III (BL16, BL18, RAJI) BL cell lines, and three LCLs (051128, 111210, and 120214). KruskalWallis tests were applied to the results of eight groups (controls and those treated with 10058-F4). Note the significant decrease of LDHA activity in all the BL lines upon inhibition of MYC transactivation ability (p = 0.0118). 26778The catalytic activity of LDHA was not changed in LCLs (encircled)levels were detected in the treated LCLs, compared with controls, in contrast with BL cell lines (Fig 7). Moreover, the control and treated LCLs, unlike BL cells, showed the same high activity of LDHA that converts pyruvate into lactate during aerobic glycolysis (Fig 8). The expression levels of the same set of genes (HK, MCT4, PDK1, HK, and LDHA) were downregulated upon inhibition of MYC transcriptional activity (Fig 7). The LDHA activity was significantly decreased in BLs treated with 10058-F4, compared with the control BL cells (Fig 8). In conclusion, we suggest that aerobic glycolysis–the Warburg effect–in BL cells is regulated by high levels of MYC, unlike LCLs where HIF1A is responsible for this phenomenon.Acute kidney injury (AKI) has been reported to be a frequent complication after orthotopic liver transplantation (LT) which is associated with poor graft survival and increased mortality [1]. The incidence of AKI after liver transplantation ranges from 20 to 64% [1, 75], and postoperative AKI is associated with the development of chronic kidney disease [10, 16]. Currently, there is no effective therapy or preventive strategy available for AKI after LT [1], although several promising strategies were investigated [179]. Therefore, identifying modifiable risk factors and preventing postoperative AKI or early intervention is essential to improve outcomes [20]. Although a number of studies have evaluated AKI after LT, the incidence and clinical risk factors are not entirely clear and evidences regarding modifiable risk factors are still lacking. This ambiguity may be explained by the variable definitions used for AKI and different clinical settings used in previous studies [1, 7]. Furthermore, most previouslyreported risk factors including longer anhepatic phase [12], intraoperative blood loss [1, 13], and large transfusion amount [3, 8, 15], model for end-stage liver disease (MELD) score [2, 7, 8, 12, 21] are not modifiable. In addition, most previous studies involved deceased donor liver transplantation and, to our knowledge, only a few groups studied the risk factors of AKI after living donor LT (LDLT) [1, 8, 13, 14]. Utsumi et al.[1] suggested the usefulness of RIFLE criteria (RIFLE = risk, injury, failure, loss, end stage) in LDLT and highlighted LDLT-specific risk factor, graft to recipient body weight ratio (GRWR). Limitations of these studies [1, 8, 13, 14] are they were performed on a relatively small number of patients over a long time period and hemodynamic or metabolic risk factors including postreperfusion syndrome [22], vasopressor infusion during operation [15], preoperative hyperuricemia [23] and intraoperative hyperglycemia [24] have not been evaluated. The purpose of this study was to determine the potentially modifiable risk factors, including hemodynamic and metabolic variables, that can identify patients at high-risk for AKI after LDLT. The second aim was to develop and validate specific clinical risk score models that accurately predict AKI after LDLT.This retrospective observational study was approved by the institutional review board of our institution (Samsung Medical Center IRB: 2013-12-080-001). We retrospectively reviewed the electronic medical records of 573 consecutive adult patients who underwent LDLT at our institution between 2007 and 2013 (Fig 1). The need for informed consent was waived given the study’s retrospective design. Patients with preoperative renal dysfunction (defined as serum creatinine>1.5 mg/dl) or renal substitute therapy (RRT) were excluded (n = 25). Clients who underwent retransplantation (n = 8) or died within 48 hrs postoperatively (n = 2) had been excluded.
