Among the genes strongly induced by hypoxia-induced transcription factors (HIFs) and highly expressed during kidney microvascular development is VEGF, which encodes a potent endothelial mitogen and chemoattractant critical for embryonic vasculogenesis and angiogenesis. In developing kidney, glomerular podocytes are particularly rich sources of VEGF, which probably serves to attract endothelial precursors into vascular clefts of immature glomeruli, promote their mitosis and differentiation into glomerular endothelial cells, and assist with maintenance of their highly differentiated state through maturation. Moreover, we have recently localized expression of HIF mRNA transcripts and protein to developing podocytes of immature glomeruli. This leads to our central hypothesis: HIF stabilization coordinates synthesis of VEGF and its receptors, and governs kidney microvascular development. To address this hypothesis, we have designed three complementary specific aims. First, we will determine whether HIFs are differentially stabilized in podocyte and endothelial cell lines exposed to different oxygen tensions. Additionally, we will selectively knockdown separate HIF alpha subunits, HIF-1 beta, and HIF gene target mRNAs, using small interfering RNA (siRNA) technologies in vitro. Second, we will determine whether kidney vascular development is affected in hybrid nephrons containing ES cells with crippled HIF activation and VEGF signaling systems. Third, we will examine in vivo consequences of losses and gains of HIF alpha subunits, using Cre-lox technology, among other approaches. These results should provide fundamentally new information on mechanisms governing renal microvessel development and maintenance, which may have broad applicability to blood vessel formation in general.
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