The overall objective of this proposal is to study the pathogenetic mechanisms involved in diabetic induced lesions in the embryonic metanephros. Three potential target genes will be examined mesodermal specific cDNA(MEST), H19, and TIN-Ag based on the spatio-temporal expression of these genes with MEST expressed in the metanephric mesenchyme H19 in the ureteric bud epithelia and TIN-Ag in an anti-polar proximal expression opposite that of H19 in the S-shaped body suggesting the three molecules are interlinked in early nephric development. Experiments are proposed under five specific aims to study the normal and abnormal biology of these molecules during renal development. I. Spatio-temporal expression of MEST, H19 and TIN-Ag during metanephric development will be investigated by using in situ hybridization, competitive PCR, immunohistochemical and immunoprecipitation methods. II. Their role in epithelial: mesenchymal interactions, prevalent during metanephrogenesis, will be assessed by antibody, antisense, transgene and transfection experiments. III. Molecular protein: protein and RNA: protein interactions will be investigated by using recombinant fusion proteins of various constructs of these three molecules. IV. Alterations in these molecules will be investigated during elevated concentration of glucose in vitro and in hyperglycemia. V. Finally, the hyperglycemia-induced/suppressed genes during embryonic renal development will be investigated using RDA and Serial Analysis of Gene Expression (SAGE).
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