Mesangial cells (MCs) subserve diverse functions and provide a scaffold that maintains the structural and functional integrity of the gomerular microvascular bed. Platelet-derived growth factor receptor beta (PDGFRb) is abundantly expressed in cultured MCs, in the injured glomerulus and in the developing kidney. Activation of this receptor stimulates migration, proliferation and differentiation of these cells. Mice deficient in PDGFRb lack MCs. The investigators first hypothesis is that a MC-specific transcription program regulates expression of the PDGFRb in these cells. Preliminary studies demonstrate that a 1.9 Kb sequence in the 5' flanking region of the PDGFRb promoter is sufficient to direct transcriptional response in MC and other cells that express PDGFRb. This sequence also contains binding sites for nuclear proteins isolated from MCs. To understand the molecular mechanisms that regulate PDGFRb gene expression, the investigators will identify and characterize the cis acting elements and transacting factors that regulate PDGFRb gene transcription in MCs and their precursors cultured from metanephric blastema. MC-specific promoter sequences will be tested in vivo to determine the spatial and temporal pattern of expression of the receptor during murine kidney development. This will be accomplished by producing and analyzing transgenic mice in which the bacterial lac Z reporter gene is placed under the control of MC-specific PDGFRb promoter sequences. The investigators second hypothesis is that specific signalling proteins play a critical role in development of MCs. Metanephric mesenchymal (MM) cells that express PDGFRb have been isolated and characterized. The investigators will determine whether a PDGFRb mutant cDNA expressing binding domain of phosphatidyl inositol kinase (PI3K) can rescue MC phenotype in vitro and in vivo. MM cells will be isolated from PDGFRb knock out mouse embryos and mutant PDGFRb cDNA expressing binding domain of PI3K will be transfected into PDGFRb-deficient MM cells. The effect of PDGF on proliferation, migration, and differentiation of these cells will be explored. The investigators will also determine the ability of PDGFRb mutants expressing PI3K binding domains to rescue MC phenotype in vivo. This will be accomplished by generation of chimeric embryos expressing the mutant PDGFRb using MC- specific promoter and studying MC phenotype in these embryos.
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