Role of NADH Dehydrogenase (Ubiquinone) 1 alpha subcomplex 4-like 2 in clear cell renal cell carcinoma

PURPOSE We delineated the functions of the HIF1α target NADH Dehydrogenase (Ubiquinone) 1 alpha subcomplex 4-like 2 (NDUFA4L2) in ccRCC and characterized NDUFA4L2 as a novel molecular target for ccRCC treatment. EXPERIMENTAL DESIGN We evaluated normal kidney and ccRCC patient microarray and RNAseq data from Oncomine and The Cancer Genome Atlas (TCGA) for NDUFA4L2 mRNA levels and the clinical implications of high NDUFA4L2 expression. Additionally, we examined normal kidney and ccRCC patient tissue samples, human ccRCC cell lines, and murine models of ccRCC for NDUFA4L2 mRNA and protein expression. Utilizing shRNA, we performed NDUFA4L2 knockdown experiments and analyzed the proliferation, clonogenicity, metabolite levels, cell structure, and autophagy in ccRCC cell lines in culture. RESULTS We found that NDUFA4L2 mRNA and protein are highly expressed in ccRCC samples but undetectable in normal kidney tissue samples, and that NDUFA4L2 mRNA expression correlates with tumor stage and lower overall survival. Additionally, we demonstrated that NDUFA4L2 is a HIF1α target in ccRCC and that NDUFA4L2 knockdown has a profound anti-proliferative effect, alters metabolic pathways, and causes major stress in cultured RCC cells. CONCLUSIONS Collectively, our data show that NDUFA4L2 is a novel molecular target for ccRCC treatment

Adjunctive rifampicin to reduce early mortality from Staphylococcus aureus bacteraemia: the ARREST RCT.

BACKGROUND: Staphylococcus aureus bacteraemia is a common and frequently fatal infection. Adjunctive rifampicin may enhance early S. aureus killing, sterilise infected foci and blood faster, and thereby reduce the risk of dissemination, metastatic infection and death. OBJECTIVES: To determine whether or not adjunctive rifampicin reduces bacteriological (microbiologically confirmed) failure/recurrence or death through 12 weeks from randomisation. Secondary objectives included evaluating the impact of rifampicin on all-cause mortality, clinically defined failure/recurrence or death, toxicity, resistance emergence, and duration of bacteraemia; and assessing the cost-effectiveness of rifampicin. DESIGN: Parallel-group, randomised (1 : 1), blinded, placebo-controlled multicentre trial. SETTING: UK NHS trust hospitals. PARTICIPANTS: Adult inpatients (≥ 18 years) with meticillin-resistant or susceptible S. aureus grown from one or more blood cultures, who had received < 96 hours of antibiotic therapy for the current infection, and without contraindications to rifampicin. INTERVENTIONS: Adjunctive rifampicin (600-900 mg/day, oral or intravenous) or placebo for 14 days in addition to standard antibiotic therapy. Investigators and patients were blinded to trial treatment. Follow-up was for 12 weeks (assessments at 3, 7, 10 and 14 days, weekly until discharge and final assessment at 12 weeks post randomisation). MAIN OUTCOME MEASURES: The primary outcome was all-cause bacteriological (microbiologically confirmed) failure/recurrence or death through 12 weeks from randomisation. RESULTS: Between December 2012 and October 2016, 758 eligible participants from 29 UK hospitals were randomised: 370 to rifampicin and 388 to placebo. The median age was 65 years [interquartile range (IQR) 50-76 years]. A total of 485 (64.0%) infections were community acquired and 132 (17.4%) were nosocomial; 47 (6.2%) were caused by meticillin-resistant S. aureus. A total of 301 (39.7%) participants had an initial deep infection focus. Standard antibiotics were given for a median of 29 days (IQR 18-45 days) and 619 (81.7%) participants received flucloxacillin. By 12 weeks, 62 out of 370 (16.8%) patients taking rifampicin versus 71 out of 388 (18.3%) participants taking the placebo experienced bacteriological (microbiologically confirmed) failure/recurrence or died [absolute risk difference -1.4%, 95% confidence interval (CI) -7.0% to 4.3%; hazard ratio 0.96, 95% CI 0.68 to 1.35; p = 0.81]. There were 4 (1.1%) and 5 (1.3%) bacteriological failures (p = 0.82) in the rifampicin and placebo groups, respectively. There were 3 (0.8%) versus 16 (4.1%) bacteriological recurrences (p = 0.01), and 55 (14.9%) versus 50 (12.9%) deaths without bacteriological failure/recurrence (p = 0.30) in the rifampicin and placebo groups, respectively. Over 12 weeks, there was no evidence of differences in clinically defined failure/recurrence/death (p = 0.84), all-cause mortality (p = 0.60), serious (p = 0.17) or grade 3/4 (p = 0.36) adverse events (AEs). However, 63 (17.0%) participants in the rifampicin group versus 39 (10.1%) participants in the placebo group experienced antibiotic or trial drug-modifying AEs (p = 0.004), and 24 (6.5%) participants in the rifampicin group versus 6 (1.5%) participants in the placebo group experienced drug-interactions (p = 0.0005). Evaluation of the costs and health-related quality-of-life impacts revealed that an episode of S. aureus bacteraemia costs an average of £12,197 over 12 weeks. Rifampicin was estimated to save 10% of episode costs (p = 0.14). After adjustment, the effect of rifampicin on total quality-adjusted life-years (QALYs) was positive (0.004 QALYs), but not statistically significant (standard error 0.004 QALYs). CONCLUSIONS: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S. aureus bacteraemia. FUTURE WORK: Given the substantial mortality, other antibiotic combinations or improved source management should be investigated. TRIAL REGISTRATIONS: Current Controlled Trials ISRCTN37666216, EudraCT 2012-000344-10 and Clinical Trials Authorisation 00316/0243/001. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 22, No. 59. See the NIHR Journals Library website for further project information.NIHR HTA Programm

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Analyses of the transcriptome and metabolome demonstrate that HIF1α mediates altered tumor metabolism in clear cell renal cell carcinoma.

Hypoxia inducible factor 1 alpha (HIF1α) is a transcription factor that is frequently stabilized and active in human clear cell renal cell carcinoma (ccRCC). We have found that constitutively active HIF1α is sufficient to cause neoplastic transformation in a murine model of ccRCC termed the TRACK model. RNA sequencing (RNAseq) and untargeted metabolomics analyses of samples from TRACK kidneys demonstrate that HIF1α activates the transcription of genes that cause increased glucose uptake, glycolysis, and lactate production, as well as a decrease in the flux of pyruvate entering the tricarboxylic acid (TCA) cycle and a decrease in oxidative phosphorylation; these changes are identical to those observed in human ccRCC samples. These studies show that a constitutively active HIF1α promotes tumorigenesis in TRACK mice by mediating a metabolic switch to aerobic glycolysis, i.e., the Warburg effect, and suggest that TRACK mice are a valid model to test novel therapies targeting metabolic changes to inhibit human ccRCC

Metabolomic analysis of key intermediates in glycolysis, lactate production, and the TCA cycle in TRACK kidneys.

<p>A: Comparison of the levels of glycolysis and lactate production intermediates, glucose, pyruvate, and lactate in TG^<b>−</b> vs. TRACK kidney samples (n = 14 and 12). □ = TG^<b>−</b>; </p><p></p><p><mi>■</mi></p><p></p> = TRACK. B: Comparison of the levels of fumarate, α-ketoglutarate, and citrate/isocitrate in WT vs. TRACK kidney samples (n = 14 and 12). □ = TG^<b>−</b>; <p></p><p><mi>■</mi></p><p></p> = TRACK. C: Schematic overview of changes to glycolysis, lactate production, and the TCA cycle in TRACK kidneys. Error bars indicate the mean ± SD. * indicates a p value <0.05, ** indicates a p value <0.01, and *** indicates a p value <0.001.<p></p

Metabolomic analysis of amino acid levels in TRACK kidneys.

<p>A: Levels of amino acids in WT vs. TRACK mouse kidneys (n = 14 and 12). □ = TG^<b>−</b>; </p><p></p><p><mi>■</mi></p><p></p> = TRACK. B: Schematic overview of changes to amino acid levels and TCA cycle. Error bars indicate the mean ± SD. * indicates a p value <0.05, ** indicates a p value <0.01, and *** indicates a p value <0.001.<p></p

Mesoscale Nanoparticles Selectively Target the Renal Proximal Tubule Epithelium

We synthesized “mesoscale” nanoparticles, approximately 400 nm in diameter, which unexpectedly localized selectively in renal proximal tubules and up to 7 times more efficiently in the kidney than other organs. Although nanoparticles typically localize in the liver and spleen, modulating their size and opsonization potential allowed for stable targeting of the kidneys through a new proposed uptake mechanism. Applying this kidney targeting strategy, we anticipate use in the treatment of renal disease and the study of renal physiology

Comparison of TRACK kidney gene transcripts with human clear cell renal cell carcinoma.

<p>Selected genes that are most highly expressed in human ccRCC, and which show increased mRNA levels (fold change > 2) in the kidney tissue of the TRACK mice relative to kidneys from wild type littermates. Human data were collected from Oncomine, and TRACK data were obtained by RNAseq.</p><p>Comparison of TRACK kidney gene transcripts with human clear cell renal cell carcinoma.</p