Knowledge about the molecular mechanisms that control development and differentiation of the renal tubule is incomplete. Ontogeny of the collecting duct is of particular importance because the two major cell types, intercalated and principal cells, are critical to maintenance of acid-base, sodium and water balance; and dysfunction of these cells likely accounts for urinary acidification, edema forming and concentrating disorders, developmental disorders of the kidney, and malignancies. We plan to study renal principal cell specific regulation of the aquaporin-2 gene, because it is specific for and essential to the function of principal cells within the collecting duct.
Our specific aims are: 1) Develop a transgenic mouse model for the In vivo study of renal principal cell specific regulatory regions within the aquaporin-2 gene. 2) Develop a cell culture model for the in vitro study of the principal cell specific regulatory regions within the aquaporin-2 gene. 3) Identify the binding sites for nuclear transcription factors within the aquaporin-2 gene that are responsible for principal cell specific expression of aquaporin-2. 4) Identify regulatory regions within the aquaporin-2 gene that collectively confer principal cell specific expression by using a combination of the in vitro cell culture model and in vivo transgenic mouse model. 5) Isolate cDNAs encoding transcription factors that are responsible for renal principal cell specific expression of the aquaporin-2 gene. These studies will utilize a combination of reporter gene studies in transgenic mice and either transfected or microinjected kidney cells, Dnase I hypersensitivity analysis, in vivo footprinting, and electrophoretic mobility shift assays to define the specific regulatory regions and nuclear protein binding sites within the aquaporin-2 gene that are responsible for renal principal cell specific expression. cDNAs encoding transcription factors that interact with these regulatory regions will be isolated using one- hybrid cloning system, or a combination of affinity purification, microsequencing and degenerate RT-PCR cloning techniques. The identification of existing or novel transcription factors involved in kidney and collecting duct specific gene expression will open the door to understanding the molecular basis for renal tubule differentiation, and will provide insight into potential disease mechanisms in the kidney.
