Generation of renin BAC transgenic mouse models for the study of regulation, lineage tracing, and tumorigenic potential of the renin-expressing cell
Glenn, Sean T.
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The objective of this work was stratified between three distinct components centralized on renin and the utilization of BAC transgenic models to study in vivo regulation, lineage-tracing as it relates to renal disease and the artificial tumorigenic potential of the renin-expressing cell in non-renal organ systems. As the rate-limiting step of the RAS, renin is fundamental to maintaining physiological homeostasis. Due to renin's importance the transcriptional regulation of the renin gene has been extensively studied, primarily in vitro using the renin expressing cell line As4.1. To verify the importance of key factors within the renin regulatory region in vivo, we introduced a GFP cassette into exon one of the renin gene contained within a 240kb BAC to create a construct that has GFP expression controlled by the renin regulatory region (RenGFP). Within the RenGFP construct we then independently deleted the enhancer, as well as mutated the Hox/Pbx site within the proximal promoter element. Transgenic animals were then created with these BAC constructs and GFP expression was analyzed throughout a spectrum of renin expressing tissues. From these studies the critical role the enhancer plays in regulating baseline expression in both kidney and submandibular gland (SMG) as well as the importance of the Hox/Pbx site to tissue specific expression of renin was validated. The RenGFP BAC transgenic model not only allows for fluorescent identification of cells that are actively expressing renin, but expression of GFP also enables the renin-expressing cell to be isolated from its in vivo context. Specifically isolating the renin-expressing cell of the developing renal vasculature has given us the ability to expression profile the purified cell population and characterize it as an activated pericyte. This characterization of the renin-expressing cell is of key importance due to current and controversial studies which have identified the pericyte of the renal vasculature as a source of myofibroblasts during the fibrotic process in the kidney. To ascertain the role that the lineal descendants of the renin-expressing pericyte population play in kidney fibrosis, homologous recombination was used to introduce a Cre-recombinase cassette into exon one of the renin gene using the same initial BAC as with our RenGFP model to create a transgenic that has Cre expression controlled by the renin regulatory region (RenCre). This transgenic can be crossed to a variety of floxed-stop-floxed reporter lines resulting in constitutive activation of the reporter in any cell that has ever expressed renin allowing us to trace the fate of the renin-expressing cell even after expression is turned off. This powerful ability to permanently tag not only every cell that has ever expressed renin but also every cell derived from the renin-expressing population allows us to trace these lineal descendants of the renin-expressing pericyte in renal disease. To do so we utilized our bigenic mice, containing both the RenCre transgene and a reporter construct, and subjected them to a model of renal fibrosis, unilateral ureter obstruction (UUO), where a ureter of one kidney is sutured shut to elicit interstitial fibrotic disease in that kidney. From these studies an increase in the number of cells of renin lineage was identified in the injured kidney. Furthermore, these cells which have expanded from renin-expressing cell origin resembled myofibroblast both in morphology and location within the interstitial space of the UUO kidney, which is not observed in healthy kidney tissue. Therefore, we propose that cells of renin origin found in the renal vasculature participate in propagating the myofibroblast population during renal disease. Beyond the ability to indefinitely tag the renin-expressing cell for lineage tracing studies in normal and disease models, the transgenic lines generated in which Cre-recombinase is driven off the Ren-1 c regulatory region (RenCre) have the valuable ability to selectively delete floxed alleles of any proposed gene specifically in the renin-expressing cell. To induce tumorigenesis within renin specific tissues, floxed alleles of p53 and Rb were selectively abrogated using our RenCre transgenic mouse. Although a number of neoplasms are observed, the primary tumor generated is a highly metastatic islet cell carcinoma of the pancreas. Further analysis using our lineage tracing abilities identifies descendants of renin-expressing cells as pancreatic alpha cells despite a lack of active renin expression in the mature pancreas. (Abstract shortened by UMI.)