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Title: Complementation of the mouse Ren-1d knockout with human renin
Author: Nelson, Robert John
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Renin is an aspartyl protease most commonly associated with its systemic role in regulation of arterial pressure and electrolyte balance through its participation in the renin-angiotensin system (RAS). Under normal physiological conditions, renin synthesis and secretion is restricted to the granulated juxtaglomerular (JG) cells of the kidney. Most mammals possess a single renin gene, however most strains of laboratory mice possess two renin genes, termed Ren-ld and Ren-2. Mice lacking a functional Ren-ld gene exhibit a complete lack of renal JG cell granulation and abnormal macula densa morphology. Transgenic mice carrying a 145 kb BAC clone encompassing the Ren-ld and Ren-2 loci show complete restoration of JG cell granulation and normal renal macula densa structure. The structural features required for formation of dense-core renin secretory granules have not yet been identified. Here we describe attempts to rescue the mouse Ren-ld knockout by complementation with a large human renin (hREN) transgene. Transgenic mice were generated using a 55 kb fragment of a PAC clone encompassing the hREN locus, with approximately 35 kb of 5'-flanking sequence. The hREN transgene was backcrossed onto the Ren-ld knockout background, and these M/TV-complemented animals demonstrated restoration of JG cell granulation in a transgene expression level-dependent manner, whilst the atypical macula densa morphology was not rescued. These experiments suggest that the human renin and mouse renin-ld proteins have conserved structural features necessary for the formation of dense-core renin secretory granules. The Ren-ld knockout mouse model provides an ideal tool for the in vivo dissection of structural features of the renin protein responsible for formation of dense-core secretory granules in JG cells. From the renal JG cells, prorenin is secreted either rapidly and intact by a constitutive pathway from the Golgi or protogranules, or is packaged into immature granules and processed into active renin, which is stored in dense-core secretory granules until its regulated release is stimulated. Evidence suggests that the product of the Ren-ld gene, renin-ld is released by the regulated pathway of secretion, whilst the product of the Ren-2 gene, renin-2, is released via the constitutive pathway. It is not known whether the renin-2 protein is able to enter the regulated pathway of secretion. To investigate the trafficking pathways of the two mouse renin proteins, fusion proteins of the renin- ld and renin-2 proteins with enhanced green fluorescent protein (EGFP) were generated. The chimaeras were expressed in vitro in the JG-like As4.1 mouse renal carcinoma cell line. Renin-ld-EGFP was localised to large granular compartments, the Golgi apparatus and also to some smaller vesicles. Renin-2-EGFP was most evident in the Golgi apparatus, with some weaker expression in the cytoplasm, with no evidence of the fusion protein in any large granular compartment. These results confirm that the mouse renin-ld and renin-2 proteins are not equivalent, and support evidence that the renin-ld and renin-2 proteins are secreted by regulated and constitutive pathways, respectively.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available