Properties of renin substrate in rabbit plasma with a note on its assay

1. Rabbit plasma enzymes that degrade angiotensin I are inhibited completely by the combination of 2,3-dimercaptopropan-1-ol (10mm), EDTA (10mm) and chlorhexidine gluconate (0.005%, w/v). These compounds do not modify the reaction of renin with renin substrate and are termed the selective inhibitors. 2. The renin substrate concentration of plasma can be measured as angiotensin I content by incubating plasma plus the selective inhibitors with renin for a time sufficient to allow complete utilization of renin substrate. 3. This reaction obeys first-order kinetics to substrate concentrations of at least 1000ng. of angiotensin I content/ml. In general, the renin substrate concentrations of normal rabbit plasmas are less than 1000ng. of angiotensin I content/ml. Thus the time required for the complete release of angiotensin I from normal plasma is inversely related to renin activity and is independent of renin substrate concentration. 4. A method for the assay of renin substrate, taking these reaction kinetics into account, is presented.     

Peptide inhibitors of converting enzyme.

Human plasma and atypical lung converting enzyme, and porcine plasma converting enzyme are substantially inhibited by other components of the renin-angiotensin system, and by angiotensin II and its analogues. Des-Asp¹ angiotensin II (angiotensin III) 0.1 mM and tridecapeptide renin substrate 0.1 mM are both effective inhibitors of human lung, plasma and porcine plasma converting enzymes. Des-Asp¹-Arg² angiotensin II also was an effective inhibitor of plasma enzymes. Bradykininase activity (kininase II) of the converting enzymes was also inhibited by angiotensin I, angiotensin III, tetradecapeptide renin substrate and tridecapeptide renin substrate. The substantial kininase and converting enzyme inhibitory effects of components of the renin-angiotensin system, suggest a potential close physiologic relationship between the kallikrein-kinin system and the renin-angiotensin system.     

An improved method for measuring angiotensin I converting enzyme activity using a highly sensitive angiotensin II radioimmunoassay

A highly sensitive assay for angiotensin I converting enzyme has been developed by using angiotensin I as a substrate. Angiotensin II generated in the reaction mixture was measured by a newly developed specific radioimmunoassay. To protect against angiotensin II destruction, bestatin, an inhibitor of renin, was also used to inhibit plasma renin activity. The reaction was stopped by adding EDTA and MK-521, inhibitors of angiotensin I converting enzyme. The specificity of the antiserum used for the angiotensin II radioimmunoassay was very high. The cross reactivity with angiotensin I was less than 0.5% and none of the proteolytic enzyme inhibitors crossreacted in the assay. The inhibitory effect of pepstatin on plasma renin activity was very high (more than 80%) under the standard assay conditions employed. Serum angiotensinase activity was completely inhibited by the addition of bestatin. An excellent correlation was obtained between this new method and the spectrophotometric method using a synthetic substrate, Hip-His-Leu. The generation of as little as 12 pM of Angiotensin II can be detected. Such low concentration have not been measurable with the usual spectrophotometric method. This new method will facilitate clinical and experimental studies on this unique enzyme, since very low levels of activity can be determined by this highly sensitive radioimmunoassay for angiotensin II.     

Kinetics of renin reaction in rat plasma (author's transl)]

Important kinetic aspects of renin reaction were studied in order to evaluate the parameters that regulate the formation rate of angiotensin I. This rate decreased throughout the incubation period of normal rat plasma and it showed a linear increase when plasma was incubated with renin-substrate. When renin was added to normal rat plasma a plateau in the angiotensin I formation rate occurred after 4-6 hours. When plasma samples containing increasing amounts of renin-substrate were incubated, the velocity of their reaction increased in proportion to the renin-substrate concentration. Under these incubation conditions, the reaction between endogenous renin and renin-substrate in normal rat plasma, proved to be a first kinetic order with respect to the substrate.     

Development of a homogeneous immunoassay for the detection of angiotensin I in plasma using AlphaLISA acceptor beads technology

Plasma renin activity (PRA) is a well-established biomarker for assessing the efficacy of various antihypertensive agents such as direct renin inhibitors, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors (ACEIs). PRA measurements are obtained through the detection and quantification of angiotensin I (Ang I) produced by the action of renin on its natural substrate angiotensinogen. The most accepted and reproducible method for PRA measurement uses an antibody capture Ang I methodology that employs specific antibodies that recognize and protect Ang I against angiotensinase activities contained in plasma. The amount of Ang I is then quantified by either radioimmunoassay (RIA) or enzyme immunoassay (EIA). In the current report, we describe the optimization of a novel homogeneous immunoassay based on the AlphaScreen technology for the detection and quantification of antibody-captured Ang I using AlphaLISA acceptor beads in buffer and in the plasma of various species (human, rat, and mouse). Ex vivo measurements of renin activity were performed using 10 μl or less of a reaction mixture, and concentrations as low as 1 nM Ang I were quantified. Titration curves obtained for the quantification of Ang I in buffer and plasma gave similar EC₅₀ values of 5.6 and 14.4 nM, respectively. Both matrices generated an equivalent dynamic range that varies from approximately 1 to 50 nM. Renin inhibitors have been successfully titrated and IC₅₀ values obtained correlated well with those obtained using EIA methodology (r² = 0.80). This assay is sensitive, robust, fast, and less tedious than measurements performed using nonhomogeneous EIA. The AlphaLISA methodology is homogeneous, does not require wash steps prior to the addition of reagents, and does not generate radioactive waste.     

Renin substrate in granules from rat kidney cortex.

1. Subcellular fractions of rat kidney cortex generated angiotensin I continuously over 2h when incubated at 37degreesC with rat renin, indicating the presence of renin substrate within cells in the renal cortex. 2. Renin substrate was located in highest specific concentration in particulate fractions. The particles containing renin substrate had a sedimentation velocity slightly lower than mitochondria and renin granules but greater than the microsomal fraction. 3. Isopycnic gradient centrifugation indicated a density of 1.190g/ml for the particles containing renin substrate, compared with 1.201 for renin granules, 1.177 for mitochondria, and 1.170 and 1.230 for lysosomes in the heavy-granule fraction. 4. In the liver, renin substrate was also found in particles, but these had a lower sedimentation rate than those from the kidney. 5. The molecular weights of renin substrate in kidney and liver granules and rat plasma were similar, namely 61000-62000. 6. On the basis of these biochemical findings, a mechanism for the intrarenal production of angiotensin, incorporating a subcellular reaction scheme, is proposed.     

Differences in the kinetic rate constants of normal and high molecular weight renin substrate from term pregnancy human plasma

We have previously reported on the differences in physical and chemical characteristics between the high-molecular weight renin substrate (HMS greater than 150,000 daltons) and the normal substrate (NMS = 60,000). In this study, the kinetic constants were determined in both HMS and NMS which were prepared by gel exclusion chromatography from the plasma of pregnant women at term. Renin substrate (angiotensinogen) levels were expressed by radioimmunoassay of angiotensin I after incubation of samples with added semi-purified human kidney renin in the presence of angiotensinase inhibitors. The kinetic constants (Km and Vmax) were determined by the method of Line-weaver-Burk plots and also the method of Wilkinson. The Km for the HMS was 1.79 micrograms angiotensin 1 equivalents ( AIeq )/ml and the Vmax = 41.2 ngAIeq /ml/h, and the Km for the NMS was 3.52 micrograms AIeq /ml and the Vmax = 138 ng/ml/h. When adding small amounts of the HMS to the NMS, the production of angiotensin I was found to increase more than that in the NMS alone. It was also observed that the renin substrate reactivities of the plasma of pregnant women, which contained small amounts of the HMS, were higher than that found in the plasma of normotensive women not taking oral contraceptives. It is suggested that the existence of small amounts of the HMS may therefore contribute to the elevation in blood pressure under the influence of estrogens.     

Could angiotensin I be produced from a renin substrate by the HIV-1 protease?

The standard angiotensin I (Ang I) radioimmunoassay for renin activity determination is a useful clinical tool for the diagnosis of high renin levels in certain cases of hypertension. It depends upon the liberation of Ang I from human plasma angiotensinogen. We considered whether a commercially available synthetic tetradecapeptide (TDP), Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, would produce authentic Ang I upon incubation with protease from human immunodeficiency virus type 1 (HIV-1). This peptide is also known to be cleaved by renin at the Leu-Leu bond to yield the decapeptide Ang I. When the TDP is incubated with the HIV-1 protease, the peptide is readily hydrolyzed. Product formation is linear with respect to time and enzyme concentration. HPLC analysis of reaction products showed two new peaks, as one would expect from the cleavage of a TDP into a decapeptide and a tetrapeptide. Amino acid analysis of HPLC-purified peaks confirmed that the HIV-1 protease cleaves TDP at the Leu10-Leu11 site to produce the desired decapeptide, Ang I. Production of Ang I by the HIV-1 protease, like human renin, is inhibited in the presence of a protease inhibitor. Implications of the discovery of an HIV-1 protease substrate that produces authentic Ang I are discussed in light of a screening assay for soluble HIV-1 protease inhibitors.     

A radiochemical renin assay

1. A synthetic 3-([(14)C]valine)-labelled tetradecapeptide renin substrate was used to measure renin concentration. Renin liberated (14)C-labelled angiotensin I, which was separated from the labelled substrate by paper chromatography. The conversion of substrate into angiotensin I was quantitated by liquid-scintillation counting of radioactivity. 2. The rate of conversion of the substrate into angiotensin I was shown to be linearly related to renin concentration and time under suitable conditions. Angiotensin generation measured in this system agrees well with that measured by bioassay. 3. It is suggested that the use of a pure substrate offers advantages that include the standardization of current units of renin measurement.     

Local renin-angiotensin systems

The existence of a local cardiovascular renin-angiotensin system (RAS) is often invoked to explain the long-term beneficial effects of RAS inhibitors in heart failure and hypertension. The implicit assumption is that all components of the RAS are synthesized in situ, so that local angiotensin II formation may occur independently of the circulating RAS. Evidence for this assumption however is lacking. The angiotensin release from isolated perfused rat hearts or hindlimbs depends on the presence of renal renin. When calculating the in vivo angiotensin production at tissue sites in humans and pigs, taking into account the extensive regional angiotensin clearance by infusing radiolabeled angiotensin I or II, it was found that angiotensin production correlated closely with plasma renin activity. Moreover, in pigs the cardiac tissue levels of renin and angiotensin were directly correlated with their respective plasma levels, and both in tissue and plasma the levels were undetectably low after nephrectomy. Similarly, rat vascular renin and angiotensin decrease to low or undetectable levels within 48 h after nephrectomy. Aortic renin has a longer half life than plasma renin, suggesting that renin may be bound by the vessel wall. In support of this assumption, both renin receptors and renin-binding proteins have been described. Like ACE, renin was enriched in a purified membrane fraction prepared from cardiac tissue. Binding of renin to cardiac or vascular membranes may therefore be part of a mechanism by which renin is taken up from plasma. It appears that the concept of a local RAS needs to be reassessed. Local angiotensin formation in heart and vessel wall does occur, but depends, at least under normal circumstances, on the uptake of renal renin from the circulation. Tissues may regulate their local angiotensin concentrations by varying the number of renin receptors and/or renin-binding proteins, the ACE level, the amount of metabolizing enzymes and the angiotensin receptor density.Abbreviations RAS renin-angiotensin system - ANG angiotensin - ACE angiotensin-converting enzyme - PRA plasma renin activity     

Measurement of renin and substrate concentrations in human serum.

Methods for the measurement of renin and renin substrate by radioimmunoassay have been described. One method of measuring renin is based on the zero-order reaction velocity of angiotensin I formation when serum is incubated with an excess of hog substrate. This method was compared with a bioassay which has been described previously (A. B. Gould, L. T. Skeggs, and J. R. Kahn, 1966, Lab. Invest.15, 1802–1813) and with another radioimmunoassay which determines renin concentration from the rate of angiotensin I formation with endogenous substrate by using the integrated form of the Michaelis-Menten equation and the kinetic constants. Similar results were obtained by these three methods when 30 samples of serum from 15 normotensive people were assayed. No evidence was found to suggest any interference by activators or inhibitors in human serum. The mean recovery of human renin added to serum in 27 experiments was 93.5 ± 10.7% (SD). In addition, the kinetic analysis of human serum showed no difference in the rate of angiotensin formation, at comparable substrate levels, in sera from normotensive people (including women taking oral contraceptives) and patients with essential hypertension.     

Purification, properties and kinetics of sheep and human renin substrates.

Sheep plasma renin substrate was purified 1,200-fold by using nephrectomised sheep plasma, followed by DEAE-Sephadex chromatography and gel filtration. The purified substrate contained 8 mu-g angiotensin II/mg protein and had an estimated molecular weight of 52,000. The kinetic characteristics of the purified substrate were identical both to those of unpurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7.5, K-m of the human renin-sheep substrate reaction was 0.29 mu-M and for sheep renin-sheep substrate, 2.0 mu-M. Sheep substrate was susceptible to peptic digestion with generation of pepsitensin. Human renin substrate was less readily purified. DEAE-Sephadex chromatography of plasma from pregnant women at 36-40 weeks' gestation produced a 70-fold increase in purity (0.9 mu-g angiotensin II/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82,000) more anionic protein than sheep substrate and was resistant to the proteolytic actions of both pepsin and sheep renin. K-m for the human renin-human substrate reaction was high and could not be accurately determined (range 3-8 mu-M, mean 5.7 mu-M). The presence of human substrate in a human renin-sheep substrate system did not alter the measured initial velocity. In both sheep and man, the normal concentration of renin substrate is considerably less than K-m and must therefore be considered a determinant of angiotensin production rate in vivo.     

Radiochemical assay for renin utilizing a synthetic insoluble substrate

In order to provide a convenient in vitro assay for renin activity, a radiolabeled renin substrate analog, N-acetyl-Asn-Arg-Val-Tyr-Ile-His-Pro-Phe-His-[³H]-Leu-Leu-Val-Tyr-Ser-Gly-Lys-Pro-OH, was prepared by solid-phase synthesis. The substrate peptide was bound covalently to agarose through the ?-amino group of its lysine residue. Incubation of this insoluble complex with partially purified hog renin resulted in the release of biologically active tritiated peptide into the soluble phase of the incubation mixture, at a rate proportional to the quantity of renin added. The optimum pH for cleavage was 6.5. The apparent K_m of the substrate was 1 × 10⁻⁴M, and the V_max was 83 pmoles tritiated peptide released/min/mg renin preparation added. The minimum amount of renin detectable by the assay was 2 μg, a quantity that would be expected to generate 1.0 pmole angiotensin per minute from the natural plasma substrate. Chymotrypsin, trypsin, papain, and pseudorenin, were also effective in cleaving labeled peptides from the insoluble substrate, but leucine aminopeptidase did not appear to release soluble radioactivity. The assay, as described, is useful for the measurement of large numbers of renin samples because of the speed and ease with which it may be performed. It is not yet sufficiently sensitive nor specific to measure the low levels of renin found in plasma.     

A comparison of the substrate specificities of cathepsin D and pseudorenin

Cathepsin D, purified from hog spleen, releases angiotensin I from tetradecapeptide renin substrate and from protein renin substrates purified from hog and human plasma. However, the enzyme does not act on the naturally occurring renin substrate as it exists in plasma nor on purified substrate in the presence of plasma. Cathepsin D releases angiotensin I quantitatively from tetradecapeptide renin substrate and does not further degrade the angiotensin I on prolonged incubation. The pH optimum for cathepsin D prolonged incubation. The pH optimum for cathepsin D acting on tetradecapeptide renin substrate is 4.5, and there is very low activity above pH 7. These properties are very similar to those of pseudorenin, an angiotensin-forming enzyme originally isolated from human kidney, indicating that cathepsin D and pseudorenin may be identical.     

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

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 renin-activatable 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.     

Renin activity determination using human plasma as a substrate

A method for human renin activity determination using human plasma as the substrate is described. Angiotensin I is generated by incubating renin with human plasma, which is available along with the commercial radioimmunoassay kit for angiotensin I. The method obviates the need to isolate and purify the substrate, human angiotensinogen, from human plasma. In addition, the assay is highly renin specific, sensitive, and convenient to use for the routine determination of active human renin during its isolation and purification from tissue extracts or from genetically engineered bacterial and nonbacterial expression systems.     

Effect of indomethacin on the adrenal renin response to nephrectomy in the rat

The role of prostaglandins in the control of adrenal renin in vivo was evaluated in nephrectomized rats. Nephrectomy increased adrenal renin from 13.2 ± 1.37 ng angiotensin I/mg protein/hr to 166.5 ± 17.3 ng angiotensin I/mg protein/hr. Indomethacin treatment significantly suppressed the adrenal renin response to nephrectomy. (47.8 ± 5.22 ng angiotensin I/mg protein/hr). Adrenal aldosterone was also suppressed by indomethacin. Adrenal prostaglandin E₂ increased after nephrectomy and decreased after indomethacin.Plasma corticosterone and serum potassium did not change after indomethacin. These data indicate that inhibition of prostaglandin synthesis by indomethacin partially blocks the adrenal renin response to nephrectomy, suggesting that prostaglandins may play a role in the adrenal response to nephrectomy.     

Measurement of renin and prorenin in cattle, hog and horse.

1. Species specific problems complicating the measurement of prorenin and renin concentrations were studied in bovine, hog and horse plasma. 2. In contrast to horse renin, bovine and hog renin reacted with rat angiotensinogen, allowing measurement of the plasma renin concentration in cattle and hog with rat angiotensinogen as exogenous substrate. 3. Trypsin treatment of plasma in order to activate prorenin generated an interfering angiotensin I immunoreactive material in all three species, most extensively in horse plasma. 4. This material could be removed in bovine and hog plasma by a cation-exchange resin, allowing an assay of the plasma prorenin concentration to be constructed in these species. 5. Another strategy has to be followed in order to measure prorenin and renin concentrations in horse plasma.     

A novel nonpeptidic, orally active renin inhibitor

BW-175 is a newly synthesized renin inhibitor which is a nonpeptidic, norleucine analog. Its IC50 values for renin activity in human, squirrel monkey, marmoset, dog, hog, rabbit and rat plasma were 3.3, 6.6, 2.4, 42, 110, 86 and 3500 nM, respectively, and 26 microM for cathepsin D. Pepsin and angiotensin converting enzyme were hardly inhibited at 10(-4) M. BW-175 showed an oral bioavailability of 2.8% at 10 mg/kg and 9.7% at 30 mg/kg in rats. In normotensive, furosemide-treated high-renin marmosets, BW-175 (30 mg/kg p.o.) caused an intensive reduction in plasma renin activity and plasma angiotensin I formation, associated with a reduction in systolic blood pressure of 10-20 mm Hg for 2 hours.     

Renin-angiotensin system, blood pressure homeostasis and renal function in galactosamine-induced fulminant hepatic failure in the guinea pig

Arterial blood pressure, renal function and plasma concentrations of renin and renin substrate (angiotensinogen) were investigated in guinea pigs subjected to galactosamine-induced (1 g/kg i.v.) liver cell necrosis. Blood pressure declined continuously by 50% during a follow-up period of 72 h which was associated with a decrease in diuresis and natriuresis to 36 and 31%, respectively. Simultaneously, plasma renin concentration increased 30-fold indicating marked reduction of renal perfusion, while plasma renin substrate concentration fell to 6% of the baseline level. There was microscopic evidence of oligemic circulatory renal damage characterized by acute proximal tubular necrosis with concomitant tubular dilatation. Short-term infusion of homologous renin substrate-enriched plasma, derived from nephrectomized animals, was followed by marked increase in mean arterial blood pressure from 34 +/- 9 to 77 +/- 7 mm Hg accompanied by marked diuresis and natriuresis. Renin substrate depletion following galactosamine-induced fulminant liver failure may represent impaired hepatic biosynthesis as well as increased renin substrate consumption due to excessive renin secretion. Angiotensinogen repletion has a beneficial effect on both renal function and blood pressure probably due to marked generation of the potent vasoconstrictor angiotensin II which consequently inhibits renin secretion. These observations strongly support the suggestion that the renin-angiotensin system is of major importance to cardiovascular homeostasis in acute liver failure.