The collecting system of the developing kidney is generated through branching morphogenesis of the ureteric bud. Approximately 1 million nephrons are induced to form as the result of as many as 20 iterations of dichotomous branching of this structure. Thus, branching morphogenesis in the developing urinary collecting system directly influences the eventual number of nephrons contained in the adult kidney. Since, reduced nephron mass may be critical determinant of both the susceptibility to and clinical course of renal disease, molecular mechanisms of branching morphogenesis are of considerable clinical and basic interest. In addition, as much as 10% of the population possess some developmental abnormality of the urinary tract with a large percentage of these involving the collecting system. MDCK and IMCD cells grown in a three-dimensional collagen matrix are ideal in vitro model systems for the identification of molecular determinants of tubulogenesis and branching in the developing kidney. When exposed to hepatocyte growth factor (HGF), MDCK cells in collagen-I gels form branching tubular structures. On the other hand, IMCD cells form branching tubular structures in response to EGF and TGF-alpha, as well as HGF. Preliminary data from the sponsor's laboratory employing both the MDCK and IMCD cell models indicates that extracellular matrix (ECM) components and matrix degrading proteinases present in the embryonic kidney are critical determinants of branching in these cells. The purpose of the work described in this proposal is to identify critical morphogenetic ECM degrading matrix metalloproteinases (MMPs), serine proteases, and tissue inhibitors of MMPs involved in branching morphogenesis of the ureteric bud. The approach will be to use a combination of substrate-SDS-PAGE analysis and Northern analysis to identify candidate morphogenetic proteinases and TIMPs which show altered expression in response to HGF, EGF, and TGF-alpha in the MDCK and IMCD cell models and which also display spatiotemporal patterns of expression in the embryonic kidney by in situ hybridization consistent with a role in formation of the collecting system. Having identified likely morphogenetic proteinases, their function will be disrupted using antisense oligonucleotides in both the cultured cell models of branching and in organ culture of the embryonic kidney and by antisense RNA producing transfection constructs in the MDCK and IMCD cell models. The Sponsor's laboratory is the ideal environment in which to carry out the work described in this proposal. The Sponsor is a recognized leader in epithelial branching morphogenesis with expertise in all of the techniques required to complete these studies. The laboratory is well equipped and the academic environment of the Renal Division at Brigham and Women's Hospital, Harvard Medical School is extremely strong in renal epithelial biology.
