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1 Department of Medicine, Division of Nephrology, University of Alabama at Birmingham, Birmingham, AL, USA; Department of Physiology and Biophysics and Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
2 Department of Medicine, Division of Nephrology, University of Alabama at Birmingham, Birmingham, AL, USA
3 High Resolution Imaging Facility, University of Alabama at Birmingham, Birmingham, AL, USA
4 Department of Physiology and Biophysics and Zilkha Neurogenetic Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
* To whom correspondence should be addressed. E-mail: petipete{at}usc.edu.
Renin release from juxtaglomerular granular cells is considered the rate-limiting step in activation of the renin-angiotensin system that helps to maintain body salt and water balance. Available assays to measure renin release are complex, indirect and work with significant internal errors. To directly visualize and study the dynamics of both the release and tissue activity of renin we isolated and perfused afferent arterioles with attached glomerulus dissected from rabbit kidney and used multiphoton fluorescence imaging. Acidotropic fluorophores, such as quinacrine and LysoTrackers, clearly and selectively labeled renin granules. Immunohistochemistry of mouse kidney with a specific renin antibody and quinacrine staining co-localized renin granules and quinacrine fluorescence. Low-salt diet for one week caused an approx. 5-fold increase in the number of both individual granules and renin positive granular cells. Time-lapse imaging showed no signs of granule trafficking or any movement, only dimming and disappearance of fluorescence from individual renin granules within 1 sec in response to 100 µM isoproterenol. There appeared to be a quantal release of the granular contents; i.e. an all-or-none phenomenon. Using As4.1 cells, a granular cell line, we observed further, classic signs of granule exocytosis, emptying of granule content associated with a flash of quinacrine fluorescence. Using a FRET-based, EDANS-conjugated renin substrate in the bath, an increase in EDANS fluorescence (renin activity) was observed around granular cells in response to isoproterenol. Fluorescence microscopy is an excellent tool to further study the mechanism, regulation and dynamics of renin release.
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