This proposal addresses the contributions of renal tubular epithelial cell interactions with the extracellular matrix in the biogenesis of renal tubules and collecting ducts. The proposed experiments focus on the alpha2beta1, and alpha1beta1, and alpha3beta1 integrins, potential collagen/laminin receptors expressed by renal epithelium. The work is built up on a foundation of previous studies that have defined the structure and function of the alpha2beta1 integrin as a cell surface collagen receptor, and very recent studies in which MDCK cells lacking the alpha2beta1 integrin as a consequence of stable overexpression of antisensense alpha2 integrin mRNA have been prepared and characterized. In contrast to parental MDCK cells and control transfectants that differentiate to form branched tubular structures when cultured in three-dimensional collagen gels in the presence of hepatocyte growth factor/scatter factor, the antisense mRNA transfectants form only large, polarized epithelial cysts with no evidence of branching or tubule formation in the morphogenetic model.
In specific aim l, we will close the experimental loop implicating that alpha2beta1 integrin in renal epithelial branching morphogenesis by rescuing the integrin- deficient phenotype by reexpression of the integrin.
Specific aim 2 will define the unique structural determinants and contributions of the alpha2 integrin cytoplasmic domain in transmitting signals necessary for tubule formation following ligation of collagen. This will be accomplished by expressing in alpha2 integrin deficient cells wild type or chimeric integrins composed of the extracellular domain of the alpha2 integrin and either no cytoplasmic domain or the cytoplasmic domains of the alpha1 or alpha3 integrins.
Specific aim 3 will explore the contribution(s) of the alpha1beta1 and/or alpha3beta1 integrin- mediated interactions with collagen to morphogenetic activity, cell polarization, and cell growth and survival by inhibiting their expression in MDCK cells with antisense mRNA.
In specific aim 4, concepts developed in the simpler MDCK cell model regarding the role(s) of integrins in renal tubulogenesis will be validated and extended by experiments in the more complex and physiologic metanephroi organ culture system using novel inhibitory reagents.
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