Highly sensitive intramolecularly quenched fluorogenic substrates for renin based on the combination of <scp>L</scp>-2-amino-3-(7-methoxy-4-coumaryl)propionic acid with 2,4-dinitrophenyl groups at various positions

Paschalidou, Katherine; Neumann, Ulf; Gerhartz, Bernd; Tzougraki, Chryssa

doi:10.1042/bj20040729

Cited by 27 publications

(33 citation statements)

References 34 publications

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“…Renin and prorenin activity was measured using a quenched fluorescence resonance energy transfer (QFRET) method that was previously described [20]. Briefly, in a 96-well plate, 15 n M of renin or prorenin were incubated in assay buffer (50 m M Tris, 100 m M NaCl, 0.002% Tween) with 5 µ M of the QFRET peptide substrate [DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His- L -Amp], where DNP is dinitrophenol, and L -Amp is L -2-amino-3-(7-methoxy-4-coumaryl)propionic acid.…”

Section: Methodsmentioning

confidence: 99%

Renin and Prorenin Activate Pathways Implicated in Organ Damage in Human Mesangial Cells Independent of Angiotensin II Production

Boie

et al. 2009

Am J Nephrol

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Background: The mechanism by which an activated renin-angiotensin system (RAS) leads to the development of renal diseases, such as fibrosis, is only partially explained by the downstream effects of angiotensin II. The discovery of a receptor that binds renin and prorenin, and the consequent production of profibrotic molecules, revealed a novel axis within the RAS pathway that may contribute to the pathogenesis of organ damage in patients with elevated renin and/or prorenin levels. Methods: To better understand the genes and networks underlying the receptor-mediated effects of renin and prorenin, a gene expression profiling study was performed on human mesangial cells in the presence of angiotensin-II-blocking agents. Results: Renin and prorenin induce highly overlapping gene expression signatures that are dependent, only in part, on the presence of the (pro)renin receptor. We found that 2 distinct pathways were activated by renin and prorenin: a TGFβ-dependent pathway and a TGFβ-independent pathway. Bioinformatic analysis was used to show that both pathways are highly enriched with genes implicated in fibrosis, hypertrophy and atherosclerosis. Conclusions: This study suggests that both renin and inactive prorenin are capable of inducing genetic programs that could contribute to end-organ damage and atherogenesis, through receptor-mediated angiotensin-independent mechanisms.

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Section: Methodsmentioning

confidence: 99%

Renin and Prorenin Activate Pathways Implicated in Organ Damage in Human Mesangial Cells Independent of Angiotensin II Production

Boie

et al. 2009

Am J Nephrol

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“…ACE and AT1R inhibitors (5, 13), AT1R blockers, and more recently renin inhibitors (31) have been used for the treatment of hypertension and attenuation of end-organ damage. In the development of these treatments, plasma renin activity (PRA) has been generally considered the most practical method for determining the activity of the RAS, using a variety of techniques, including immunoassays (3), fluorescence (FRET) substrates (24,27), mass spectrometry (8), and magnetic sensors (34). In preclinical studies, these technologies have been limited to plasma assessment.…”

mentioning

confidence: 99%

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

Zhang

Preda

Vasquez

et al. 2012

American Journal of Physiology-Renal Physiology

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. A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo. Am J Physiol Renal Physiol 303: F593-F603, 2012. First published June 6, 2012; doi:10.1152/ajprenal.00361.2011.-The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a reninactivatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments. optical tomography; low-salt diet; hypertension THE RENIN-ANGIOTENSIN SYSTEM (RAS) plays an important role in the regulation of blood volume, electrolyte homeostasis, and systemic vascular resistance, which together affect cardiac output and arterial pressure (11). Renin, a highly specific aspartyl protease, is primarily stored and released from juxtaglomerular (JG) cells within the afferent arterioles of the kidney glomeruli and plays an important regulatory role in RAS function. JG cells can sense a reduction in afferent arteriole pressure in the kidney leading to direct release of renin, and specialized adjacent cells (the macula densa) can also trigger JG renin release upon sensing of decreased plasma sodium chloride in renal tubules or changes in kidney sympathetic nerve signaling. Release of renin into the blood then mediates the first and rate-limiting step of the RAS cascade by cleaving angiotensinogen to generate angiotensin I (ANG I). ANG I is further cleaved by angiotensin-converting enzyme (ACE) to generate bioactive angiotensin II (ANG II), which binds to angiotensin recepto...

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“…13 We chose the rst 15 N-terminal amino acids of angiotensinogen (DRVYIHPFHLVIHNE) for detecting the activity of human renin. It should be noted that there is no photophysical constraint on the length of the sequence, in contrast to e.g.…”

Section: Design Of Renin Substratesmentioning

confidence: 99%

“…based on HPLC-electrospray-tandem mass spectrometry, 7 uorogenic substrates 8-10 or FRET. [11][12][13] For uorogenic substrates, however, a uorophore must be placed next to the cleavage site, and FRET substrates are only quenched efficiently if the distance between the donor and acceptor is typically in the range of 2 to 6 nm.…”

Section: Introductionmentioning

confidence: 99%

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Kinetic analysis of renin and its inhibitors by detecting double-labelled peptidic substrates with an immunoassay

Gorris

2013

Analyst

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The proteolytic activity of renin is a key element in the regulation of blood pressure and a main target for inhibitor design. Currently, the activity of renin and its inhibitors is mainly analyzed using radioimmunoassays or FRET-substrates, which both have their limitations. Here, a novel kinetic assay is presented that combines the advantages of a homogeneous proteolytic reaction and a robust heterogeneous detection in a sandwich immunoassay format. The proteolysis in solution is not influenced by surface interactions and yields accurate kinetic values, while the specific detection of the cleavage products on a microtiter plate strongly reduces interference by concomitant substances and allows for a self-referenced signal readout. A new enzyme kinetic scheme for the inhibition of renin has been developed and validated by using the model inhibitor pepstatin. This kinetic analysis is amenable to parallelization for large-scale inhibitor screening. Furthermore, it can be easily adapted to inhibitors of other medically important proteases.

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Highly sensitive intramolecularly quenched fluorogenic substrates for renin based on the combination of L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid with 2,4-dinitrophenyl groups at various positions

Cited by 27 publications

References 34 publications

Renin and Prorenin Activate Pathways Implicated in Organ Damage in Human Mesangial Cells Independent of Angiotensin II Production

Renin and Prorenin Activate Pathways Implicated in Organ Damage in Human Mesangial Cells Independent of Angiotensin II Production

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

Kinetic analysis of renin and its inhibitors by detecting double-labelled peptidic substrates with an immunoassay

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