Glomerular capillary development involves initial ingrowth of capillary sprouts into primitive S-shaped bodies. Capillary sprouts derive either from vessels invading the developing kidney, or from vascular structures formed in situ. Initial endothelial cell proliferation and sprouting is followed by cell cycle arrest, cell flattening and fenestration. Mechanisms guiding glomerular capillary development are highly complex, involving cell-matrix and cell-cell interactions. This proposal aims to explore mechanisms whereby mediators present in the developing kidney and produced by renal epithelial cells undergoing morphogenesis influence glomerular endothelial cell capillary formation, directed tissue invasion and eventual differentiation. Insofar as normal glomerular capillary development is a prerequisite for normal glomerular function, the work is relevant to renal disease. The involvement of TGF-beta together with two endothelial cell mitogens, namely basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF) in these processes will be explored. Their prior evidence shows that in vitro capillary formation and apoptosis are associated events and require TGF-beta mediated signals in glomerular endothelial cells. They now plan to determine whether the process of apoptosis is necessary for capillary formation, or whether it occurs only in those cells which do not have the requisite signals either from matrix and/or from the mitogens to form capillaries. They will explore whether cells protected against programmed cell death will form capillaries (instead of undergoing apoptosis) in response to TGF-beta, and to what extent protection against apoptosis is afforded by mitogens and matrix attachment. In addition to TGF-beta, they specifically plan to explore the involvement of bFGF and VEGF in the processes of capillary formation and fenestration. Extensive use will be made of glomerular capillary endothelial cells stably transfected with appropriate transdominant negative mutant receptors for appropriate growth regulators to be studied. The in vitro model systems involve a simple capillary format ion assay, collagen gel invasion assay, and co-culture with epithelial cells undergoing morphogenesis themselves. The in vivo model system involves transfection and/or transduction of post-birth developing rat kidney with the appropriate dominant negative receptor construct followed by morphological examination of glomerular capillaries at various time points during the period of ongoing glomerulogenesis.
