Chronic Kidney Disease progression : from molecular pathways to urinary biomarkers

par Mordi Muorah

Thèse de doctorat en Biologie cellulaire et moléculaire

Sous la direction de Fabiola Terzi.

  • Résumé

    Chronic Kidney Disease (CKD) and its progression has become an important public health issue owing to its strong links to cardiovascular morbidity and mortality. To understand molecular pathways implicated the process of CKD progression is several-fold useful. It can enable drug development inhibiting identified pathways. It provides us with potentially measurable molecules for patient categorisation and treatment. In this translational medicine project, we move from the bench, understanding the role played by signal transducer and activator of transcription 3 (STAT3) to the bedside, measuring and identifying urinary molecules in patients all in the context of CKD progression. Thus we identified potential downstream effectors of STAT3 in CKD progression: lipocalin 2, TIMP1 and PDGFB. To achieve this we combined an approach of in vivo, in silico and in vitro methods. We observed on immunohistochemistry the activation (phosphorylation) specifically of tubular STAT3, in mice sensitive to subtotal nephrectomy (Nx) prior to their development of fibrosis and confirmed that genetic deletion of STAT3 in the tubules protected the mice kidneys. Exploiting microarray data, from remnant kidneys of mice with different susceptibilities to Nx, and combining this with in silico data of potential STAT3 DNA binding sites (DBS) provided genes differentially regulated by STAT3. In the upregulated genes, in which the DBS were conserved in 4 or more species, we identified and confirmed on RT-PCR (both in the remnant kidneys and in a mouse cell line and independently in IMCD3 cells), genes regulated by deletion of STAT3. We specifically studied secreted factors along the hypothesis of a crosstalk between renal tubular cells with activated STAT3 inducing secreted factors to activate nearby resident fibroblasts to secrete collagen and thus induce fibrosis. In parallel, we examined molecules known to be involved in the pathological processes of CKD progression. We rigorously validated the use of 16 commercialised ELISA kits in the urine following strict industry criteria and excluded 14 failing to meet the criteria in a pilot study of 75 subjects. We next took advantage of a well-characterised cohort of 229 patients with CKD with different rates of progression, as determined by serially measured glomerular filtration rates (mGFRs). We measured by the validated ELISA kits the molecules in the urine collected at baseline visit. We identified a combination of urinary biomarkers that can predict fast progression (i.e. degradation in mGFR at > 10% baseline mGFR/ year) after taking into account demographic risk factors for progression and albuminuria. The three biomarkers identified were EGF, MCP1 and TGF-α.

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