title: Bimodal dynamics of granular organelles in primary renin-expressing cells revealed using TIRF microscopy.
abstract: Renin is the initiator and rate-limiting factor in the renin-angiotensin blood pressure regulation system. Although renin is not exclusively produced in the kidney, in nonmurine species the synthesis and secretion of the active circulatory enzyme is confined almost exclusively to the dense core granules of juxtaglomerular (JG) cells, where prorenin is processed and stored for release via a regulated pathway. Despite its importance, the structural organization and regulation of granules within these cells is not well understood, in part due to the difficulty in culturing primary JG cells in vitro and the lack of appropriate cell lines. We have streamlined the isolation and culture of primary renin-expressing cells suitable for high-speed, high-resolution live imaging using a Percoll gradient-based procedure to purify cells from RenGFP+transgenic mice. Fibronectin-coated glass coverslips proved optimal for the adhesion of renin-expressing cells and facilitated live cell imaging at the plasma membrane of primary renin cells using total internal reflection fluorescence microscopy (TIRFM). To obtain quantitative data on intracellular function, we stained mixed granule and lysosome populations with Lysotracker Red and stimulated cells using 100 nM isoproterenol. Analysis of membrane-proximal acidic granular organelle dynamics and behavior within renin-expressing cells revealed the existence of two populations of granular organelles with distinct functional responses following isoproterenol stimulation. The application of high-resolution techniques for imaging JG and other specialized kidney cells provides new opportunities for investigating renal cell biology.
authors.affiliation: BHF/University Centre for Cardiovascular Science, University of Edinburgh, Queen's Medical Research Institute, Edinburgh, United Kingdom; cbuckley@exseed.ed.ac.uk.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Buckley
authors.affiliation: Edinburgh Super Resolution Imaging Consortium, Heriot-Watt University, Riccarton Campus, Edinburgh, United Kingdom.
authors.forename: Alison R
authors.initials: AR
authors.lastname: Dun
authors.affiliation: BHF/University Centre for Cardiovascular Science, University of Edinburgh, Queen's Medical Research Institute, Edinburgh, United Kingdom.
authors.forename: Audrey
authors.initials: A
authors.lastname: Peter
authors.affiliation: Department of Pathology, Royal Infirmary of Edinburgh, Edinburgh, United Kingdom; and.
authors.forename: Christopher
authors.initials: C
authors.lastname: Bellamy
authors.affiliation: Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, New York.
authors.forename: Kenneth W
authors.initials: KW
authors.lastname: Gross
authors.affiliation: Edinburgh Super Resolution Imaging Consortium, Heriot-Watt University, Riccarton Campus, Edinburgh, United Kingdom.
authors.forename: Rory R
authors.initials: RR
authors.lastname: Duncan
authors.affiliation: BHF/University Centre for Cardiovascular Science, University of Edinburgh, Queen's Medical Research Institute, Edinburgh, United Kingdom.
authors.forename: John J
authors.initials: JJ
authors.lastname: Mullins
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000378:metabolism*; D007606:Juxtaglomerular Apparatus / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008853:Microscopy / Q000379:methods; D012083:Renin / Q000378:metabolism*; D012084:Renin-Angiotensin System / Q000502:physiology*
chemical_list: D012083:Renin
keywords: TIRFM; granule tracking; juxtaglomerular; renin