Real-time imaging of renin release in vitro

Abstract

lease from juxtaglomerular granular cells is considered the ratelimiting 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 glomeruli dissected from rabbit kidneys 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 colocalized renin granules and quinacrine fluorescence. A low-salt diet for 1 wk caused an approximately fivefold 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 the dimming and disappearance of fluorescence from individual renin granules within 1 s in response to 100 M 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, the emptying of granule content associated with a flash of quinacrine fluorescence. Using a fluorescence resonance energy transfer-based, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (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 for the further study of the mechanism, regulation, and dynamics of renin release. multiphoton excitation; fluorescence microscopy; juxtaglomerular apparatus; renin activity; quinacrine THE JUXTAGLOMERULAR APPARATUS (JGA) represents a major structural component of the renin-angiotensin system (RAS) and is one of the most important regulatory sites of renal salt and water conservation and blood pressure maintenance. Release of renin from juxtaglomerular granular cells in the terminal afferent arteriole is considered the rate-limiting step of RAS activation and is precisely controlled by several mechanisms (16,21,24). Reductions in renal perfusion pressure, activation of the sympathetic nervous system, local hormones, and reductions in macula densa salt transport result in increases in circulating and interstitial renin levels that lead to enhanced generation of ANG I with subsequent conversion to ANG II (23,24). ANG II, one of the most potent hypertensive autacoids and the major product of the RAS, exerts its effects on renal vascular and tubular structures, as well as on other organs.

Olfactory receptor responding to gut microbiota-derived signals plays a role in renin secretion and blood pressure regulation

“…4B). Consistent with this result, plasma renin measurements (27,28) revealed that Olfr78 −/− mice fed a standard chow diet rich in plant polysaccharides have significantly lower plasma renin levels than wild-type mice fed the same diet ( Fig. 4C, P < 0.05).…”

“…The disappearance of quinacrine over time was measured as an index of renin release (26,27). Addition of 10 mM propionate to the bathing solution of JGA/glomeruli from wild-type mice resulted in a decrease in cell-associated quinacrine fluorescence.…”

“…Propionate (P) induces quinacrine release (which is a surrogate for renin release) in wild-type but not Olfr78 −/− animals (B). PRA levels (plasma renin activity, normalized ex vivo to wild-type and determined as the slope of the time course of fluorescent product production in a kinetic assay) (27,28) are significantly reduced in Olfr78 −/− mice (C). * indicates statistical significance.…”

New insights into the renoprotective actions of the renin inhibitor aliskiren in experimental renal disease

“…Moreover, the implications from this work and the autoradiographic data are that aliskiren may provide an unusual example in which an inhibitor incorporates into its (still intracellular) target even before the latter is secreted. In this case, (pro)renin would be secreted from JG cells to the plasma or the tubulo-interstitium 103 in an already inhibited state. The renin activity in this (pro)renin-aliskiren complex would be inhibited: this renin (or prorenin, which may undergo activation at tissue sites) could not cleave Aogen, and the tissue and systemic RAAS cascades would not be activated.…”

Intrarenal renin–angiotensin system

“…Multiphoton florescence imaging has recently made it possible to visualize and study the dynamics of both the release and tissue activity of renin in isolated and perfused afferent arterioles with attached glomeruli dissected from rabbit kidneys. Supportive of renin release into the interstitium, was the observation that quinacrine-labeled renin-granules disappeared while there was a simultaneous increase in renin activity of fluid around granular cells of the M a n u s c r i p t perfused afferent arteriole (Peti-Peterdi et al, 2004. In addition, under most circumstances interstitial renin concentration is much higher than that of the circulating blood.…”