Chronic Kidney Disease (CKD) is a major burden of public health affecting millions of people around the world. Even though many aspects of the complex mechanisms orchestrating progression of renal disease have been identified, so far there is no specific treatment to slow down or prevent CKD progression. Therefore, identifying novel specific therapeutic targets and proposing more effective treatments against the progression of CKD is one of the major challenges of public health today. Our preliminary data showed that genetic deletion of a protein named Celen (we can't disclose the real name in the general public because is under Inserm patent deposit), exacerbated the decline of renal function in the Ang II- and nephrotoxic serum (NTS)- induced models of CKD. Inversely, activation of Celen protects against oxidative stressors (enhanced at the early stage of CKD) and prevents increase of apoptotic markers in renal cells. The specific hypothesis behind this proposal, strongly backed-up by our preliminary data, is that activation of Celen will counteract the deleterious effects of chronic renal inflammation and fibrosis, and preserve renal structure and function during kidney diseases providing a novel therapeutic target against CKD. The objectives of the present proposal are to: 1) Evaluate the role of Celen in the progression of experimental kidney disease (NTS model) using cell-specific Celen (podocyte or endothelial) gene deletion. 2) Dissect the molecular pathways implicated in the signaling of Celen in the kidney and identify novel regulators in the progression of glomerulonephritis. Specifically, we will investigate the role of Celen in mitochondrial function (i.e mPTP opening, Ca2+ overload). Using RNA-Seq and ChIP-Seq/-qPCR technologies, we will identify the full range of Celen target genes and the epigenetic enzymes mediating its nuclear signaling. 3) Examine whether cell-specific overexpression of Celen (in podocytes or endothelial cells depending on the results of 1) in adult mice is sufficient to halt or reverse the progression of glomerulonephritis. 4) Determine if pharmacological activation of Celen is nephroprotective. 5) Explore the translational relevance of its expression in biopsies. Partner's 2 lab is in charge of a cohort (CORIRLA) of CKD patients at Tenon hospital, Paris. The proposed task is to explore the cellular localization of Celen and its downstream effectors in different types of glomerulonephritis. This is a pilot study. If the results are confirmed, they will pave the way for a wider study involving more patients and partners which will be the subject of a future financial demand. We have therefore assembled two groups of internationally distinguished investigators to continue an existing collaboration and conduct basic and translational kidney research. Both partners have complementary expertise: Partner 1 is a renowned expert of the signaling, pharmacology and disease mechanisms involving Celen and Partner 2 has an international recognized expertise in renal pathophysiology and experimental models of kidney disease along with platforms for renal hemodynamics and in vivo imaging. After 3 years, we expect to deliver the following results: 1) Establish Celen as a novel actor in CKD ; 2) Obtain an in-depth analysis of Celen signaling in nephroprotection ; 3) Demonstrate that pharmacological intervention on Celen slows down or arrests progression of CKD ; 4) Observational study of Celen expression and signaling pathways in selected human nephropathies. The impact of our proposal will be scientific by identifying a new pathophysiological mechanism of CKD, socioeconomic by providing a proof of concept of a new therapy approach for CKD and cultural by giving the opportunity to young fellows to receive a high quality training thus improving their capacity for further career development in the academic or private sector.

CKD is common affecting 10-12% of the adult population, and 30% or more over 70 years of age. Cardiovascular disease (CVD) is the leading cause of death in CKD patients with steadily increased risk as kidney function declines up to 10-20 times higher in end-stage renal disease (ESRD) than in the general population. CKD is mainly associated with two types of CVD: accelerated atherosclerosis and specific CKD-related CVD including arteriosclerosis and cardiac abnormalities (i.e. left ventricular hypertrophy and diastolic dysfunction). CVD risk assessment is currently based upon the Framingham risk score and traditional risk factors (i.e. diabetes, hypertension, dyslipidemia, and smoking), which underestimate CKD-related CV complications. Additional CKD-related factors called uremic toxins may ameliorate this prediction in CKD patients. However, in front of the multiplicity of known and unknown uremic toxins, global methods such as proteomic are necessary to confirm and to explore this issue. Therefore, we propose to build on those observations and to validate and develop molecular profiles predicting CV complications (i.e. the main objective of PROTEOMARK_CKD) and CKD progression (i.e. the secondary objective) in the large-scale CKD-REIN prospective cohort. Indeed, CKD-REIN (Chronic Kidney Disease-Renal Epidemiology and Information Network) is a large prospective cohort study carried out in 40 nephrology outpatient facilities, nationally representative with respect to geography and public or private legal status. From July 2013 through March 2016, 3033 adult patients (> 18 years) with moderate (stage 3, eGFR within 45-59 mL/min/1.73 m2) or advanced (stage 4, eGFR 15-44 mL/min/1.73 m2) CKD, without prior chronic dialysis or kidney transplantation, gave informed consent to participate in the study and were included during a routine nephrology visit. In these patients, clinical events and deaths will be collected from baseline through December 31, 2018. At this time, almost all patients will have completed 3-year follow-up. For each cardiovascular event, whether fatal or non-fatal, clinical research associates are trained to document events by up-loading medical or death reports in the CKD-REIN information system. Primary coding of clinical events and deaths considered in this project will be performed by a physician who will ensure that CV events are sufficiently documented for adjudication. Baseline biological sampling for urinary proteome analyses will be available at the biobank .We will use a case-cohort design to select participants for the urinary proteome analysis. In such a design, all incident cases as defined above over the period from baseline through December 31, 2018 and a random sample of the overall population at baseline are included. The PROTEOMARK-CKD consortium is composed of two highly complementary teams providing clinical (Ziad Massy, INSERM-U1018), epidemiologic and statistical (Bénédicte Stengel, and Marie Metzger INSERM-U1018), clinical proteomics (Joost Schanstra, Inserm U1048) and bioinformatic (Julie Klein, Inserm U1048) expertise and operational capacity which form the pillars of the project. PROTEOMARK_CKD will identify and validate molecular signatures that allow stratifying CKD patients at risk for CV complications by combining established clinical characteristics with proteomic profiles derived from plasma and urine, reflecting the underlying pathogenic processes. Once validated, these biomarkers will be introduced in clinical practice. The present program will significantly improve our understanding of the mechanisms of CKD associated CVD and CKD progression. The validation of CKD related factors may lead to the discovery of new pathophysiological pathways and allow a "pharmacophenomics" approach, associating biomarker-guided treatment strategies and individually targeted pharmacological treatments, then to be tested in patients with CVD or progression in RCTs.

DDysregulation of fat metabolism in liver and adipose tissues is a hallmark of insulin resistance. White and brite/beige adipose tissues, and liver produce proteins and lipids with systemic action on insulin sensitivity. The regulation of the pathways involved in the synthesis, release and use of fatty acids shall influence the secretory capacities of these organs to promote systemic effects on insulin sensitivity. Survey of human data shows that fatty acids are not the only metabolic villains in insulin resistance. There are major gaps in the understanding of the impact of modified fat metabolism in adipocytes and hepatocytes on insulin sensitivity. Therefore, HepAdialogue aims at identifying new mediators of insulin sensitivity which production is influenced by key nodes of regulation in adipose and hepatic lipid metabolic pathways. The partners have identified three proteins that play such a role. In white adipocytes, hormone-sensitive lipase (HSL) independently of its role in adipose tissue lipolysis physically interacts with the glucose-responsive transcription factor, ChREBP, to control de novo lipogenesis and fat cell insulin signaling. ChREBP also influences insulin sensitivity through its major role in the control of de novo fatty acid synthesis in the liver. The nuclear receptor PPARalpha controls fat oxidation. In adipose tissue, it also promotes white-to-brite conversion of fat cells. In the liver, PPARalpha acts as a free fatty acid sensor during adipose tissue lipolysis and controls ketogenesis. Unexpectedly, PPARalpha shares a common transcriptional target with ChREBP, the hepatokine FGF21 which controls brown and white adipose tissue metabolism. Identification of lipids and proteins with putative endocrine action has been initiated by the Partners. In HepAdialogue, it will be completed though combined lipidomics, proteomics and transcriptomics analyses of adipocytes and hepatocytes in vitro and, of fat and liver from adipose-specific and hepatocyte-specific knock out mice in vivo. This part of the work is supported by already established models and preliminary unpublished data. Following validation on complementary models of a short list of secreted lipid and protein species, the influence of adipose factors on hepatocyte glucose metabolism and insulin signaling will be investigated. Conversely, we will study the influence of hepatic factors on adipocyte glucose metabolism and insulin signaling. Regulation of the production of the novel endocrine factors will be investigated in preclinical models of type 2 diabetes and tissue samples from human cohorts. Various pharmacological and nutritional approaches known to impact on the metabolic nodes of lipid metabolism will be used to manipulate the production and levels of the identified factors. Upon project completion, we aim at having identified and validated new secreted molecules with confirmed bioactivity in cell culture. Seeking industrial partnership, the next step will be to test whether the compounds themselves, their precursors or some inhibitors may have the capacity to reverse insulin resistance and associated metabolic disorders and/or to induce browning of white adipose tissues in vivo.

The incidence of obesity worldwide has increased drastically during last decades, consequently obesity and related disorders now constitute a very concerning international public health issue. The World Health Organization estimates that over 1,4 billion adults are in overweight and of these, 500 millions are obese. Obesity can be lethal as it involves a wide range of additional health problems including diabetes, chronic kidney diseases, cardiovascular diseases or fatty liver related dysfunctions. The dimension of this future public health problem is unseen, and on top of education (diet + physical activity), we have no choice than trying to find therapeutic tools to help to take over this up-coming flow of patients. In this context, preliminary findings in the lab, and published reports show a certain protein called HMGB1 (High Mobility Group B1), as very intriguing protein, mainly localized in the nucleus and able to regulate gene transcription in all tissues but also capable of initiating and maintaining a potent inflammatory reaction once released in the extracellular space. Blood and tissue (adipose tissue and liver) levels of HMGB1 are increased in obese patients and obese mice compare to normal conditions suggesting that HMGB1 could play an important role in obesity progression and related pathologies including fatty liver. To test this hypothesis, we will study the role of HMGB1 protein, in a holistic manner, with a translational project combining mice preclinical models, and human patients cohort displaying a various degree of liver disorders called NASH. In mice, HMGB1 role in liver physiology during fatty liver occurrence will be addressed by ablating hmgb1 gene in hepatocytes specifically (cre-lox). In parallel, hmgb1 gene polymorphisms (SNP) will be studied in patients and then correlate with the NASH severity. Then we will functionally validate identified SNP(s), in vitro on a human hepatocyte cell line, using innovating gene editing technology. We will also use this opportunity (having human blood collection) to measure HMGB1 circulating levels and evaluate whether it could be considered as a relevant NASH biomarker. Finally, biological function of circulating HMGB1 during obesity will be addressed by using a specific pharmacological inhibitor, known to particularly block extracellular HMGB1. On top of this, hmgb1 gene will be deleted in adipocytes and macrophages specifically, in order to better understand whether these cell types could contribute to HMGB1 circulating pool, and unravel which precise role HMGB1 could have in these key cell types during metabolic stress. In conclusion, this very innovating and ambitious project will allow us to better understand HMGB1 biological function as a nuclear factor and extracellular molecule during obesity and related diseases. This project will also help to potentially establish HMGB1 as a relevant and attractive pharmacological target in humans, to treat obesity and/or peripheral tissue impairments.

A major public health issue comes from the increasing needs of platelet concentrates to be transfused in older adults, in individuals under anti-cancer therapy, or in bone marrow graft receivers during the recovery period. Despite the implementation of safety procedures, presence of unsuspected pathogens and undesirable post-transfusion reactions represent objective risks. Today, platelets can be generated in vitro, but after years of competition, the yields are still far from being compatible with a cost effective alternative to transfusion. Platelets are generated in the bone marrow through two series of key processes, (i) in the bone marrow, progenitor cells progressively differentiate into fully mature MKs, which (ii) cross the endothelial barrier of the sinuses to release cellular fragments that will disrupt into functional platelets in the blood stream. The goal of the project is to decipher how the development of megakaryocytic cells is positively influenced by the stroma, namely the extracellular matrix, mesenchymal and endothelial cells, a prerequisite before implementing in vitro mass platelet production. The project is based on a number of recent observations from the already collaborating 2 partners, (Partner 1: UMR-S949, H. de La Salle; Partner 2: UMR1048, F. Gaits-Iacovoni), published or not, and related to the two above mentioned series of events. (i) They have unraveled that in vitro embryonic stromal cells favor the development of MK precursor/progenitor cells from progenitor cells. These observations open new/complementary strategies to specify the major steps occurring during MK differentiation, at the level of hematopoietic precursors as well as of supporting cells, including endothelial cells. (ii) They recently demonstrated that in vitro mesenchymal stromal cells favor the development of “thrombocytogenic” MKs, displaying a high competence to produce platelets. This effect could be recapitulated by inhibiting the aryl hydrocarbon receptor (AhR). We now want to decipher the biochemical and genetic events associated to this unique in vitro pathway and to define which in vivo processes these events represent. (iii) They recently unraveled how in vitro cytoskeleton dynamics control the differentiation of MKs, their contacts with extracellular matrices and the involvement of podosomes or related structures and, (iv) began an ultrastructural analysis of the in vivo contact between MKs and sinuses. These later investigations need to be correlated in order to clarify which dynamic membrane mechanisms are associated to the crossing of the endothelial barrier by MKs and the subsequent generation of platelets. Technically, our project is based on complementary and synergic approaches, using advanced imaging techniques routinely used by the two partners (super-resolution microscopy, Focused Ion Beam Transmission electron microscopy, correlative light electron microscopy) together with classical complementary tools (RNA-seq gene profiling, biochemical analysis of signal transduction, genetic engineering, and use of specific cellular effector agonists or antagonists). Our main attention will be focused on human biological systems, using in vitro approaches, although complementary investigations will require in vivo explorations using genetically deficient mice. This project will bring a new fine cognitive view about the biology and generation of MKs and platelets, both in vivo and in vitro. We expect to unravel novel strategies that will unlock the limitations of in vitro platelet production.