2004 Global Researcher Conference Proceeding

April 09 - 11, 2004

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Conference: 2004 Global Researcher Conference
Title: Collecting Duct Specific Gene Regulation: Creation And Use of Transgenic Mouse Models
Authors: Miller, R. Lance; Kohan, M.D., Donald; Breton, Sylvie; Brown, Dennis; Nelson, Raoul
Institutions: Massachusetts General Hospital and Harvard Medical School, Massachusetts General Hospital, University of Utah Health Sciences Center, University of Utah
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Nelson

My lab is interested in improving our understanding of development and differentiation of the collecting duct of the mammalian nephron. The mature collecting duct consists of the principal and intercalated cells, which are essential to the maintenance of water, sodium and acid base-balance. The differentiation of the ureteric bud into the mature collecting duct requires the expression of specific genes required for the function of these cell types. Collecting duct specific gene expression is likely determined by many transcription factors acting together. In order to better understand the regulation of gene expression in the collecting duct we have developed transgenic mouse models expressing green fluorescence protein (GFP) in principal and intercalated cells. We first developed transgenic mice expressing green fluorescent protein in either principal. The mouse AQP2 promoter was used to drive expression of GFP in transgenic mice. Detail analysis by RT-PCR of organs and immunofluorescence microscopy demonstrated kidney and principal cell-specific expression. Fluorescence microscopic analysis of embryos demonstrated that expression of GFP occurred at E 18 when AQP2 is expressed. We next developed transgenic mice expressing green fluorescent protein in intercalated cells. The V-ATPase B1 subunit promoter was used to drive expression of GFP in transgenic mice. Detail analysis by real-time RT-PCR of organs and immunofluorescence microscopy demonstrated kidney and intercalated cell-specific expression of green fluorescent protein. Fluorescence microscopic analysis of embryos demonstrated GFP expression at E 19. These transgenic mouse studies demonstrate that principal and intercalated cell specific expression is determined by cell specific transcription in the collecting duct of kidney and that developmental timing of expression is late in gestation when the collecting duct begins to function.

We are now using these transgenic mice expressing green fluorescent protein to better understand collecting duct specific gene expression. We have developed a fluorescence assisted microdissection technique to isolate collecting ducts from whole kidney, and cortex, outer medulla and inner medulla. In addition, we have developed fluorescence activated cells sorting to isolate intercalated cells from whole kidney and principal cells from primary cultures of kidney. Our collaborators are using laser capture microdissection to isolate principal and intercalated cells from kidney sections. These cell isolation techniques have been combined with real-time RT-PCR to determine the gene expression patterns in principal and intercalated cells of the collecting duct.

We are now using these transgenic mice and cell isolation techniques to identify transcription factors involved in principal cell specific gene expression. Principal cells were isolated from primary cultures of AQP2-EGFP transgenic kidneys. RNA and cDNA from these cells was used to perform suppressive subtractive hybridization. This identified two families of transcription factors, including the epithelial Ets transcription factors. Real-time RT-PCR analysis demonstrated that these are highly expressed in kidney compared to other organs and in collecting ducts compared to other tubules, and in both principal and/or intercalated cells. Studies are underway to determine if these factors are involved in collecting duct specific gene regulation. The potential exists that these factors may be involved in collecting duct differentiation and collecting duct diseases.

The principal cells within the collecting duct are responsible for nephrogenic diabetes insipidus. The X-linked form of the disease is due to mutations in the V2 receptor for vasopressin thus leading to impaired vasopressin signaling and downregulation of aquaporin-2 expression. One potential pharmacological approach to the disease is to design pharmacologic agents that target vasopressin independent signaling pathways via transcription factors that modulate aquaporin-2 gene expression.

Nelson and the researchers working with him are investigating the collecting ducts (CD) within the kidney. More specifically, they want to better understand what regulates the genes that will be expressed in the kidney CD and mechanics of how this happens. To help them do this, the researchers developed two different lines of transgenic mice that expressed a green fluorescence protein (GFP) in either principal or intercalated cells of the CD. Their work showed the coming into being of the principal and intercalated cells by the process of cell specific transcription in the kidney CD. Further, the expression of these cells occurs only late in gestation when the CD begins to function.

Using the mice and analytical techniques they developed, the researchers were also able to identify two families of transcription factors that are found in both principal and intercalated cells which were more highly expressed in the kidney and kidney CDs compared to other organs and tubules. (The CD is a tubule, i.e., a tiny tube.) Nelson, et al., are now investigating whether or not these factors are important to CD specific gene regulation. Their work may prove helpful in the development of NDI therapies that can influence AQP2 expression without having to go through the AVP/V2R molecular signaling pathway.