Chez la souris, la néphrogenèse débute par l'apparition du blastème metanéphrogène à 9.5 dpc. Une transition mésenchymo-épithéliale, comportant 5 étapes, débute a 11.5 dpc et aboutit au rein mature, composé de 3 structures : glomérules, tubules, et capillaires glomérulaires. Les étapes initiales du développement rénal peuvent être récapitulées en culture ex vivo; toutefois, l'organogenèse terminale et la maturation rénale sont incomplètes, et les structures rénales obtenues ex vivo ne sont pas fonctionnelles. Une étude du développement vasculaire in vivo au cours du développement rénal montre une angiogenèse (cellules Pecam-1 positives) et une vasculogenèse (cellules VEGFR-1 positives) précoces, dès 10.5 dpc. Une analyse quantitative par qRT-PCR confirme le rôle de Hif1α et VEGF dans la vasculogenèse rénale. En outre, la voie PGC1α, inductrice de VEGF indépendante de HIF, est activée en conditions hypoxiques. Pour améliorer le développement vasculaire rénal ex vivo, nous proposons un modèle de culture avec micro-perfusion rénale. L'étude morphologique par immunofluorescence des reins après culture micro-perfusée montre une survie tissulaire normale (TUNEL), et une intégrité anatomique (Néphrine, Cytokératine, WT1), en particulier vasculaire (Pecam-1). Une perfusion de vivo-morpholinos WT1 aboutit à une perte d'expression de WT1, confirmant le caractère fonctionnel de notre modèle. En conclusion, nous montrons le rôle précoce de l'angiogenèse et de la vasculogenèse au cours du développement rénal ; nous identifions le rôle de PGC1α dans la vasculogenèse rénale en conditions hypoxiques, et nous proposons une nouvelle technique de culture rénale ex vivo. / In mice, nephrogenesis starts with the formation of the metanephric mesenchyme, at e9.5 dpc. A mesenchymal epithelial transition, consisting of 5 steps, starts at e11.5 dpc, and leads to a mature kidney, composed of 3 main structures: glomeruli, tubules, and capillaries. The initial steps of renal development can be recapitulated ex vivo; however, terminal organogenesis and maturation are impaired, and the explants are not functional. A study of vascular development in vivo during renal development shows that angiogenesis (Pecam-1 positive cells) and vasculogenesis (VEGF-R1 positive cells) occur early, at e10.5 dpc. A quantitative analysis, by qRT-PCR, shows that Hif1α and VEGF play a major role in renal vasculogenesis. Moreover, the PGC1α signaling pathway, a HIF independent VEGF inductor, is activated under hypoxic conditions. To improve ex vivo vascular development, we propose a novel culture technique, with micro-perfusion of the explant. A morphologic analysis of the kidneys obtained by micro-perfused cultures shows no apoptosis (TUNEL), a conserved parenchymal structure (Nephrin, Cytokeratin, WT1), and a proper vascular development (Pecam-1). A micro-perfusion of WT1 vivo-morpholinos leads to a decrease in WT1 expression, thus validating our model. In conclusion, we showed the early role of angiogenesis and vasculogenesis in renal development, we analyzed PGC1α role in hypoxic kidney cultures, and we proposed a novel kidney culture model.

PKC βII is alternatively spliced during acute insulin stimulation in L6 skeletal muscle cells. This PKC βII isoform is critical in propagating GLUT4 translocation. PKC β protein and promoter dysfunction correlate with human insulin resistance. TZD treatment ameliorates whole-body insulin-resistance. Its primary target is adipocyte PPAR γ, which it activates upon binding. This causes both altered circulating serum FFA concentrations and adipokine secretion profile. How TZDs affect the intracellular signaling of skeletal muscle cells is unknown. RT-PCR and Western blot analysis showed that TZDs elevated PKC βII by a process that involves co-transcriptional splicing. PGC1 α overexpression most closely resembled TZD treatment by increasing PKCβII protein levels and keeping PKC βI levels relatively constant. Use of a heterologous PKCβ promoter driven PKC β minigene demonstrated that PPARγ could regulate the PKCβ promoter, but whether this is direct or indirect is unclear. SRp40 splicing factor has been shown to dock onto the PGC1 α CTD and influence splicing. SRp40, through overexpression and silencing, appears to play a part in PKC β promoter regulation. PKC β promoter regulation was also studied in 3T3-L1 cells. TZDs were experimentally shown to have no role in PKC β promoter regulation despite PPARγ activation. Chromatin immunoprecipitation assays revealed PU.1 as a putative PKC β transcription factor that can cross-talk with the spliceosome, possibly through SRp40 which was also associated with the PKC β promoter. 3T3-L1 adipocyte differentiation revealed a novel developmentally-regulated switch from PKC βI to PKCβ II, using western blot and Real-Time PCR analysis. Pharmacological inhibition of PKC β II using CGP53353 and LY379196 blocked [ 3 H]2-deoxyglucose uptake and revealed a functional role for PKC β II in adipocyte ISGT. CGP53353 specifically inhibited phosphorylation of PKC β II Serine 660 and not other critical upstream components of the insulin signaling pathway. Subcellular fractionation and PM sheet assay pointed to PKC β II-mediated regulation of GLUT4 translocation to the PM. Co-immunoprecipitation between PKC β II and GLUT4 allude to possible direct interaction. Western blot and immunofluorescence assays show PKC β II activity is linked with Akt Serine 473 phosphorylation, thus full Akt activity. Western blot and co-immunoprecipitation suggested that insulin caused active mTORC2 to directly activate PKC βII. Data support a model whereby PKCβ II is downstream of mTORC2 yet upstream of Akt, thereby regulating GLUT4 translocation.