ReninSense680 FAST
In Vivo Fluorescent Imaging Agent

Part #
Agent Description

11079

ReninSense 680 FASTTM including 1 vial, 10 doses (based on a 20-25g mouse) - Fluorescence agent specifically activated by renin activity within the renin-angiotensin system pathway (RAS). Optically silent in the unactivated state, becoming highly fluorescent when activated.

ReninSense680 FAST is a member of a family of activatable fluorescent imaging agents comprising a novel architecture, termed F.A.S.T. (Fluorescent Activatable Sensor Technology), that confers an improved pharmacokinetic profile with a broader range of imaging timepoints. This architecture also offers higher target specific signal with reduced background.

ReninSense680 FAST is a Renin activatable agent that is optically silent upon injection and produces fluorescent signal after cleavage by renin produced in the kidneys. The Renin-Angiotensin System (RAS) is the hormone system involved in regulating blood pressure and fluid balance in the body.  ReninSense680 FAST is recognized and activated by rodent forms of renin in preclinical animal models. ReninSense680 FAST may be used to monitor abnormal RAS function, progression of disease and the efficacy of therapeutic treatment in disorders such as hypertension and cardiovascular disease and some neurological diseases.

Chemistry Overview

ReninSense680 FAST is a VivoTag-S 680 fluorescently-labeled mouse/rat renin-specific activatable agent. The agent comprises a pharmacokinetic modifier to minimize non-specific kidney signal and increase blood half-life.

ReninSense680 FAST is stable as a dry solid stored at 2°-8°C for 3 months and as an aqueous solution stored at 2°-8°C  for 7 days.
 Schematic diagram of ReninSense680 FAST

Protease Selectivity Panel (24 hrs.)

ReninSense680 FAST (1 mM final concentration in the assay) was cleaved in the presence of 0.1 mM recombinant human (rhRenin), rat renin (rrRenin), and mouse renin (rmRenin), human neutrophil elastase (HNeu Elastase), human neutrophil cathepsin G (HNeu CatG), human liver cathepsin D (HLiver Cat D), trypsin or plasmin. Reactions were carried out in optimal buffers, pH and temperature. Fluorescence was measured using a fluorescence microplate fluorimeter at 24 hrs after beginning the reaction.

ReninSense680 FAST is efficiently activated by  mouse and rat renin but not by human renin. Other human enzymes (reported to cleave angiotensinogen sequences) also cleave the agent. The agent is relatively stable in normal mouse plasma as illustrated in the diagram on left.

Stability in Plasma

1 µM of ReninSense680 FAST agent was incubated in normal mouse plasma (4x diluted in assay buffer: 1xPBS, pH 7.4, 1 mM EDTA) at 37°C for 24h. Fluorescence was determined by fluoremeter readings. Cleavage products were analyzed by HPLC.

ReninSense680 FAST is stable in normal mouse plasma with no apparent
de-quenching and/or cleavage (activation by rmRenin shown for comparison).  The results are confirmed by HPLC @ 24 h time point as illustrated in the diagram on right.

Plasma Half-Life

Normal C57BL/6 mice were injected i.v. with 2 nmol of agent (n = 2-3 mice/time point) and blood drawn (in EDTA) at different times thereafter. Blood was spun at 17,000 rpm for 10 min at 4°C and plasma samples (50  µL) treated with cold methanol (150 µL); the resulting samples were mixed and spun at 12,000 rpm for 6 min and the supernatant (100 µL) analyzed by RP-HPLC using absorbance detection (675 nm). A standard curve was created starting at a concentration of ReninSense680 FAST of 2.5 µM and diluting 2 fold.

ReninSense680 FAST shows a plasma half-life of ~ 6 hours.

Low Salt Diet Model Characterization

Kidney renin activity in C57BL/6 mice was increased experimentally by feeding mice a low salt diet (0.02% sodium) and including a diuretic (amiloride - 0.1 mg/5 mL or ~5 mg/kg/day) in their drinking water for 48h**.

Mice fed a regular chow diet (0.1% sodium) and water were used as controls. After 2 days of diet, plasma and kidneys were collected.

Plasma was diluted in PBS, pre-incubated with or without the specific rodent renin inhibitor L-810 (300 nM), at RT for 30 min. ReninSense680 FAST was added (1 µM final concentration) incubated at 37°C for various times and fluorescence read in a microplate fluorimeter.

Ten micron kidney frozen sections were incubated with ReninSense680 FAST (1 µM final concentration), at 37 °C for 3 h and imaged using fluorescence microscopy

In Vitro: Low Salt Diet Model, Plasma Renin
ReninSense680 FAST is activated by plasma from mice with low salt diet, activation is  blocked by the renin inhibitor L-810 (Panel A). Plasma renin upregulation (increased kidney renin release) is confirmed by a competition-based enzyme immunoassay to quantify angiotensin I resulting from angiotensinogen cleavage by plasma renin (Panel B) (Analytical Biochemistry 388 (2009) 134-139).

 In Vivo: Renin Upregulation in Kidneys

24h after starting C57BL/6 mice on normal or low salt diet, ReninSense680 FAST was  injected i.v. (2 nmol/mouse) and mice imaged at 48 h (24h post-agent). Immediately after, kidneys were removed and imaged in reflectance mode. A. Images of representative mice using FMT (upper panels) and excised kidneys imaged in fluorescence reflectance mode (FRI, lower panels). B. Measurement of kidney region fluorescence by FMT 2500 LX (left panel) and average fluorescence intensity of removed kidneys (right panel).  Data represents a  composite of 2 separate studies (n=4).

Low sodium diet significantly increases ReninSense680 FAST signal in kidneys as shown by FMT imaging in vivo and reflectance imaging ex vivo.

 ReninSense680 FAST:  Validation of Renin Imaging with a Selective Renin Inhibitor

The selectivity of renin imaging by ReninSense680 FAST was established by L-810 inhibitor studies.

Ex Vivo Validation of LSD: A Model of Renin Upregulation

Ex vivo renin imaging agent ReninSense680 FAST shows increased renin activity in tissue and plasma samples after LSD/A treatment that is inhibited by L-810

ReninSense 680 FAST Properties & Specifications
1.  Absorbance and fluorescence maxima of ReninSense680
FAST in 1x PBS.

2.  As determined by RP-HPLC and measuring absorbance at
675 nm.

Normalized absorbance (grey), excitation (blue) and fluorescence emission (red) spectra of ReninSense680 FAST in 1x PBS.

Material  (Needs to be reconstituted)

ReninSense680 FAST: Each vial contains 24 nmol of ReninSense680 FAST in dry solid form. ReninSense680 FAST has been filtered through a 0.2 µm filter prior to drying. Reconstitute ReninSense680 FAST with 1.2 mL of 1 x PBS before injecting into animals. The packaged material provides sufficient reagent for imaging approximately 10 mice (weighing ~25 grams each) when using the recommended dose of 2 nmol (100 µL) of ReninSense680 FAST per mouse.

Properties

The physical characteristics of ReninSense680 FAST can be found in table above.

Storage and Handling

Upon receipt, ReninSense680 FAST should be STORED AT 2-8 oC AND PROTECTED FROM LIGHT.

When stored and handled properly, ReninSense680 FAST is stable for up to 3 months in dry solid form.

Before opening the vial check to ensure that all of the solid material is at the bottom of the vial.

After reconstituting with PBS, gently swirl the solution to ensure that the solid is fully in solution.

Once reconstituted with 1 x PBS, the solution is stable up to 7 days when stored at 2-8 ºC and protected from light.

In Vivo Imaging and Applications

Renin is a peptide hormone secreted by the kidney from granular cells of the juxtaglomerular apparatus and is the first step in a cascade of events that lead to increased blood pressure.  Renin is upregulated in response to decrease in arterial blood pressure, decrease in plasma sodium chloride levels, or sympathetic nervous system activity. Renin cleaves angiotensinogen (produced in the liver) to yield angiotensin I, which is further converted to angiotensin II by angiotensin-converting enzyme (ACE).   Angiotensin II constricts blood vessels, increases aldosterone secretion from the adrenals, and stimulates a thirst reflex, each leading to an increase in blood pressure.  Kidney imaging of ReninSense680 FAST can detect 3- to 4-fold increases in kidney renin activity in response to 2-3 days of low salt diet in C57BL/6 mice. 

The generally recommended procedure for in vivo imaging with ReninSense680 FAST is administration via intravenous injection and imaging 24 hours post injection. Earlier and later timepoints may be appropriate for some disease models, and the optimal imaging timepoint for any application should be determined empirically.

ReninSense680 FAST enables imaging of Renin activity in applications including:

cardiovascular disease,

    certain models of impaired renal function,

      chronic hyperthyroidism,

      hypertension, and

      some neurological diseases.

      Part #
      Agent Description

      11079

      ReninSense 680 FASTTM including 1 vial, 10 doses (based on a 20-25g mouse) - Fluorescence agent specifically activated by renin activity within the renin-angiotensin system pathway (RAS). Optically silent in the unactivated state, becoming highly fluorescent when activated.