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Establishing an in vitro model of olfactory receptor 78‐mediated renin release
Author(s) -
Poll Brian,
Pluznick Jennifer
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb352
Subject(s) - receptor , renin–angiotensin system , immunofluorescence , intracellular , in vitro , agonist , microbiology and biotechnology , cell , chemistry , endocrinology , cell culture , in vivo , medicine , biology , biochemistry , antibody , immunology , genetics , blood pressure
Recent studies by our laboratory have identified Olfactory Receptor 78 (Olfr78) as a short chain fatty acid (SCFA) receptor which modulates blood pressure. We previously reported that in ex vivo preparations of glomeruli, Olfr78 responds to SCFA's such as propionate to mediate renin release in the juxtaglomerular apparatus (JGA) in the kidney. Similarly, whole‐animal Olfr78 KO mice have lowered plasma renin levels, and are hypotensive.(Pluznick, Protzko et al. 2013). While it is known that Olfr78 is a G‐protein coupled receptor, the precise signaling pathway linking SCFA binding and renin release is not known. In this study, we aimed to establish a cell model which could recapitulate Olfr78‐mediated renin release. To do this, we utilized As4.1 cells, a juxtaglomerular cell line. We found that this cell line does not natively express Olfr78 by RT‐PCR; therefore we studied As4.1 cells either with or without stable expression of a Lucy‐ FLagRho‐Olfr78 construct. (Lucy and Rho tags have been previously shown by our group and others to promote the stable ectopic expression of olfactory receptors). As4.1 release renin upon treatment with relevant stimuli, including the beta‐agonist isoproterenol. We first established methods by which we can detect and quantify this renin release including immunofluorescence, live‐cell imaging, and media fluorescence. As4.1 cells treated with isoproterenol (100μM, 20min) showed a decrease in intracellular renin staining via immunofluorescence as quantified by ImageJ (−35%, n=3, p<0.05). Live cell imaging with quinacrine, an acidotrophic dye that naturally accumulates in renin‐containing granules, also shows a decrease in the fluorescence of these granules after treatment with 100μM isoproterenol (20 min, 30% decrease, n=3, p<0.05). Additionally, we were able to detect renin release by measuring quinacrine fluorescence in the media after treatment of cells with isoproterenol. Media from As4.1 cells had a fluorescence increase from a relative baseline of 1.00 +/− 0.02 in untreated samples to 1.18 +/− 0.02 after isoproterenol treatment (100uM, 10 min, n=8 p<0.05). After establishing these methods, we tested whether we could also quantify renin release in As4.1 cells that stably express Olfr78 following propionate treatment. Media from cells expressing Olfr78 had a fluorescence increase from a relative baseline of 1.00 +/− 0.02 in untreated samples to 1.21+/−0.023 after treatment with propionate (20mM, 10 min, n=8, p<0.05) as compared to wild‐type AS4.1 cells 1.02+/−0.02 (n=8, p>0.05). We hypothesize that Olfr78‐mediated renin release requires increases in cAMP levels, and that this release is inhibited by the elevation of cytosolic calcium levels. Efforts are underway to test this hypothesis and determine the essential downstream components of Olfr78‐ mediated renin release in this cell model, which will provide insights into renin release and blood pressure regulation in vivo. Support or Funding Information Support: NIDDK This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .