Transcription factor NFE2L1 decreases in glomerulonephropathies after podocyte damage

Funding: This work was supported by NHS Lothian. This project received funding from the European Union’s Horizon 2020 research and innovation programme under Grant Agreement No. 101017453 as part of the KATY project, and from NuCana plc.Podocyte cellular injury and detachment from glomerular capillaries constitute a critical factor contributing to kidney disease. Notably, transcription factors are instrumental in maintaining podocyte differentiation and homeostasis. This study explores the hitherto uninvestigated expression of Nuclear Factor Erythroid 2-related Factor 1 (NFE2L1) in podocytes. We evaluated the podocyte expression of NFE2L1, Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2), and NAD(P)H:quinone Oxidoreductase (NQO1) in 127 human glomerular disease biopsies using multiplexed immunofluorescence and image analysis. We found that both NFE2L1 and NQO1 expressions were significantly diminished across all observed renal diseases. Furthermore, we exposed human immortalized podocytes and ex vivo kidney slices to Puromycin Aminonucleoside (PAN) and characterized the NFE2L1 protein isoform expression. PAN treatment led to a reduction in the nuclear expression of NFE2L1 in ex vivo kidney slices and podocytes.Publisher PDFPeer reviewe

YAP translocation precedes cytoskeletal rearrangement in podocyte stress response : a podometric investigation of diabetic nephropathy

KH was funded by a University of St Andrews 600th Anniversary Ph.D. scholarship. ME and DH were supported by NHS Lothian.Podocyte loss plays a pivotal role in the pathogenesis of glomerular disease. However, the mechanisms underlying podocyte damage and loss remain poorly understood. Although detachment of viable cells has been documented in experimental Diabetic Nephropathy, correlations between reduced podocyte density and disease severity have not yet been established. YAP, a mechanosensing protein, has recently been shown to correlate with glomerular disease progression, however, the underlying mechanism has yet to be fully elucidated. In this study, we sought to document podocyte density in Diabetic Nephropathy using an amended podometric methodology, and to investigate the interplay between YAP and cytoskeletal integrity during podocyte injury. Podocyte density was quantified using TLE4 and GLEPP1 multiplexed immunofluorescence. Fourteen Diabetic Nephropathy cases were analyzed for both podocyte density and cytoplasmic translocation of YAP via automated image analysis. We demonstrate a significant decrease in podocyte density in Grade III/IV cases (124.5 per 106 μm3) relative to Grade I/II cases (226 per 106 μm3) (Student’s t-test, p<0.001), and further show that YAP translocation precedes cytoskeletal rearrangement following injury. Based on these findings we hypothesize that a significant decrease in podocyte density in late grade Diabetic Nephropathy may be explained by early cytoplasmic translocation of YAP.Publisher PDFPeer reviewe

Podocyte injury elicits loss and recovery of cellular forces

K.E. Haley was funded by a University of St. Andrews 600th Anniversary PhD Scholarship. M.C.G. acknowledges support by the Human Frontiers Science Program (RGY0074/2013), the Scottish Funding Council (via SUPA), EPSRC (EP/P030017/1) and the ERC Starting Grant ABLASE (640012). D.J.H. was supported by NHS Lothian. M.C.G. and P.A.R. acknowledge support by the BBSRC (BB/P027148/1).In the healthy kidney, specialized cells called podocytes form a sophisticated blood filtration apparatus that allows excretion of wastes and excess fluid from the blood while preventing loss of proteins such as albumin. To operate effectively, this filter is under substantial hydrostatic mechanical pressure. Given their function, it is expected that the ability to apply mechanical force is crucial to the survival of podocytes. However, to date podocyte mechanobiology remains poorly understood, largely due to a lack of experimental data on the forces involved. Herein, we perform quantitative, continuous, non-disruptive and high-resolution measurement of the forces exerted by differentiated podocytes in real time using a recently introduced functional imaging modality for continuous force mapping. Using an accepted model for podocyte injury, we find that injured podocytes experience near complete loss of cellular force transmission, but that this is reversible under certain conditions. The observed changes in force correlate with F-actin rearrangement and reduced expression of podocyte-specific proteins. By introducing robust and high-throughput mechanical phenotyping and by demonstrating the significance of mechanical forces in podocyte injury, this research paves the way to a new level of understanding of the kidney. In addition, we integrate cellular force measurements with immunofluorescence and perform continuous long-term force measurements of a cell population, which has not been feasible with established force mapping techniques. As such, our approach has general applicability to a wide range of biomedical questions involving mechanical forces.Publisher PDFPeer reviewe

Tissue-specific immunopathology in fatal COVID-19

Funding: Inflammation in COVID-19: Exploration of Critical Aspects of Pathogenesis (ICECAP) receives funding and support from the Chief Scientist Office (RapidResearch in COVID-19 programme [RARC-19] funding call, “Inflammation in Covid-19: Exploration of Critical Aspects of Pathogenesis; COV/EDI/20/10” to D.A.D., C.D.L., C.D.R., J.K.B., and D.J.H.), LifeArc (through the University of Edinburgh STOPCOVID funding award to K.D., D.A.D., and C.D.L.), UK Research and Innovation (UKRI) (Coronavirus Disease [COVID-19] Rapid Response Initiative; MR/V028790/1 to C.D.L., D.A.D., and J.A.H.), and Medical Research Scotland (CVG-1722-2020 to D.A.D., C.D.L., C.D.R., J.K.B., and D.J.H.). C.D.L. is funded by a Wellcome Trust Clinical Career Development Fellowship(206566/Z/17/Z). J.K.B. and C.D.R. are supported by the Medical Research Council (grant MC_PC_19059) as part of the International Severe AcuteRespiratory Infection Consortium Coronavirus Clinical Characterisation Consortium (ISARIC-4C). D.J.H., I.H.U., and M.E. are supported by the Industrial Centre for Artificial Intelligence Research in Digital Diagnostics. S.P. is supported by Kidney Research UK, and G.T. is supported by the Melville Trust for the Cure and Care of Cancer. Identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and sequencing work was supported by theU.S. Food and Drug Administration grant HHSF223201510104C (“Ebola Virus Disease: correlates of protection, determinants of outcome and clinicalmanagement”; amended to incorporate urgent COVID-19 studies) and contract 75F40120C00085 (“Characterization of severe coronavirus infection inhumans and model systems for medical countermeasure development and evaluation”; awarded to J.A.H.). J.A.H. is also funded by the Centre of Excellence in Infectious Diseases Research and the Alder Hey Charity. R.P.-R. is directly supported by the Medical Research Council Discovery Medicine North Doctoral Training Partnership. The group of J.A.H. is supported by the National Institute for Health Research Health Protection Research Unit in Emerging and Zoonotic Infections at the University of Liverpool in partnership with Public Health England and in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.Rationale: In life-threatening Covid-19, corticosteroids reduce mortality, suggesting that immune responses have a causal role in death. Whether this deleterious inflammation is primarily a direct reaction to the presence of SARS-CoV-2 or an independent immunopathologic process is unknown. Objectives: To determine SARS-CoV-2 organotropism and organ-specific inflammatory responses, and the relationships between viral presence, inflammation, and organ injury. Methods: Tissue was acquired from eleven detailed post-mortem examinations. SARS-CoV-2 organotropism was mapped by multiplex PCR and sequencing, with cellular resolution achieved by in situ viral spike protein detection. Histological evidence of inflammation was quantified from 37 anatomical sites, and the pulmonary immune response characterized by multiplex immunofluorescence. Measurements and main results: Multiple aberrant immune responses in fatal Covid-19 were found, principally involving the lung and reticuloendothelial system, and these were not clearly topologically associated with the virus. Inflammation and organ dysfunction did not map to the tissue and cellular distribution of SARS-CoV-2 RNA and protein, both between and within tissues. An arteritis was identified in the lung, which was further characterised as a monocyte/myeloid-rich vasculitis, and occurred along with an influx of macrophage/monocyte-lineage cells into the pulmonary parenchyma. In addition, stereotyped abnormal reticulo-endothelial responses, including excessive reactive plasmacytosis and iron-laden macrophages, were present and dissociated from viral presence in lymphoid tissues. Conclusions: Tissue-specific immunopathology occurs in Covid-19, implicating a significant component of immune-mediated, virus-independent immunopathology as a primary mechanism in severe disease. Our data highlight novel immunopathological mechanisms, and validate ongoing and future efforts to therapeutically target aberrant macrophage and plasma cell responses as well as promoting pathogen tolerance in Covid-19.Publisher PDFPeer reviewe

Spatial analysis of NQO1 in non-small cell lung cancer shows its expression is independent of NRF1 and NRF2 in the tumor microenvironment

This work was funded in part by NuCana plc, Lothian NHS and the Industrial Centre for AI Research in Digital Diagnostics (iCAIRD), which is funded by Innovate UK on behalf of UK Research and Innovation (UKRI) [project number: 104690].Nuclear factor erythroid 2-related factor 1 (NFE2L1, NRF1) and nuclear factor erythroid 2-related factor 2 (NFE2L2, NRF2) are distinct oxidative stress response transcription factors, both of which have been shown to perform cytoprotective functions, modulating cell stress response and homeostasis. NAD(P)H:quinone oxidoreductase (NQO1) is a mutual downstream antioxidant gene target that catalyzes the two-electron reduction of an array of substrates, protecting against reactive oxygen species (ROS) generation. NQO1 is upregulated in non-small cell lung cancer (NSCLC) and is proposed as a predictive biomarker and therapeutic target. Antioxidant protein expression of immune cells within the NSCLC tumor microenvironment (TME) remains undetermined and may affect immune cell effector functions and survival outcomes. Multiplex immunofluorescence was performed to examine the co-localization of NQO1, NRF1 and NRF2 within the tumor and TME of 162 chemotherapy-naïve, early-stage NSCLC patients treated by primary surgical resection. This study demonstrates that NQO1 protein expression is high in normal, tumor-adjacent tissue and that NQO1 expression varies depending on the cell type. Inter and intra-patient heterogenous NQO1 expression was observed in lung cancer. Co-expression analysis showed NQO1 is independent of NRF1 and NRF2 in tumors. Density-based co-expression analysis demonstrated NRF1 and NRF2 double-positive expression in cancer cells is associated with improved overall survival.Publisher PDFPeer reviewe

NUC-7738 regulates β-catenin signalling resulting in reduced proliferation and self-renewal of AML cells

This study was supported by research funding from NuCana plc to all authors.Acute myeloid leukemia (AML) stem cells are required for the initiation and maintenance of the disease. Activation of the Wnt/β-catenin pathway is required for the survival and development of AML leukaemia stem cells (LSCs) and therefore, targeting β-catenin is a potential therapeutic strategy. NUC-7738, a phosphoramidate transformation of 3’-deoxyadenosine (3’-dA) monophosphate, is specifically designed to generate the active anti-cancer metabolite 3’-deoxyadenosine triphosphate (3’-dATP) intracellularly, bypassing key limitations of breakdown, transport, and activation. NUC-7738 is currently in a Phase I/II clinical study for the treatment of patients with advanced solid tumors. Protein expression and immunophenotypic profiling revealed that NUC-7738 caused apoptosis in AML cell lines through reducing PI3K-p110α, phosphorylated Akt (Ser473) and phosphorylated GSK3β (Ser9) resulting in reduced β-catenin, c-Myc and CD44 expression. NUC-7738 reduced β-catenin nuclear expression in AML cells. NUC-7738 also decreased the percentage of CD34+ CD38- CD123+ (LSC-like cells) from 81% to 47% and reduced the total number and size of leukemic colonies. These results indicate that therapeutic targeting of the PI3K/Akt/GSK3β axis can inhibit β-catenin signalling, resulting in reduced clonogenicity and eventual apoptosis of AML cells.PreprintPublisher PDFPeer reviewe

NUC-7738 induces apoptosis in AML cell lines.

OCI-AML3 and U937 cells were treated with NUC-7738 at 5, 10 and 20 μM for 48 hrs and apoptosis was determined by Annexin-V DAPI staining. (A) dot plots generated during flow cytometry analysis highlighting the percentage of live (LL), early apoptotic (LR), late apoptotic (UR) and dead (UL) cells. (B) Bar graphs highlight the proportion of cells which are viable, in early apoptosis or in late apoptosis as determined by Annexin-V / DAPI staining, and analysis by flow cytometry. The percentages shown indicate the proportion of total events within each of the respective categories as gated on the flow cytometry plots. Gating strategy highlighted in S1 Fig in S1 File. Each bar represents the mean percentage from three independent experiments with error bars indicating SD showing NUC-7738 caused a reduction in viable cells and an increase in early and late apoptotic cells in all cells examined. NUC-7738 did not induce apoptosis in the KG1a cell line (S20 Fig in S1 File).</p

Fig 3 -

NUC-7738 reduces colony formation and LSC-like cells in leukemia (A) HL-60 cells were treated with NUC-7738 for 72 hours followed by plating 30 x 103 cells onto MethoCult medium and recording colonies after 14 days. The total number and size of colonies were reduced following NUC-7738 treatment (B) KG-1a cells were treated with 5, 10 and 20 μM NUC-7738 for 48 hours followed by cell surface staining for CD34, CD38 and CD123, in addition to DAPI staining to negate dead cells. These data are from three independent experiments. Each bar denotes mean ± SD * P<0.05, ** P<0.01.</p

S1 File -

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A schematic model illustrating the proposed role of NUC-7738 in suppressing β-catenin in leukemia cells.

A schematic model illustrating the proposed role of NUC-7738 in suppressing β-catenin in leukemia cells.</p