A pepstatin-immunometric assay for active renin in human plasma

1988 ◽  
Vol 174 (3) ◽  
pp. 345-349
Author(s):  
Jitsuo Higaki ◽  
Toshio Ogihara ◽  
Masaru Nishiura ◽  
Kazuo Murakami ◽  
Yuichi Kumahara
1979 ◽  
Vol 57 (s5) ◽  
pp. 89s-92s ◽  
Author(s):  
F. H. M. Derkx ◽  
B. N. Bouma ◽  
H. L. Tan-Tjiong ◽  
M. A. D. H. Schalekamp

1. Human plasma was treated at 4°C with acid, trypsin, plasmin, streptokinase, urokinase, active Hageman factor fragment (β-XIIa) and β-XIIa-activated plasma prekallikrein (Fletcher factor). The conversion of inactive into active renin (activation) was studied in normal plasma (n = 10), Hageman factor-deficient plasma (n = 2), Fletcher factor-deficient plasma (n = 1) and plasminogen-free plasma (n = 4). 2. In normal plasma inactive renin was activated at pH 7·5 after treatment at pH < 4·0; at pH 3·3 the results were the same as with trypsin. This was also the case in plasminogen-free plasma. In Hageman factor-deficient plasma and in Fletcher factor-deficient plasma, however, the quantities of renin that were activated after acidification were much smaller than with trypsin. The addition of physiological amounts of active kallikrein to pH 3·3-pretreated Hageman factor-deficient plasma caused complete activation of renin. In contrast, the addition of active Hageman factor fragment to pH 3·3-pretreated Fletcher factor-deficient plasma had little or no effect. 3. Plasmin, streptokinase-activated plasminogen and urokinase-activated plasminogen activated inactive renin in pH 4·0-pretreated normal plasma as well as in pH 4·0-pretreated Hageman factor-deficient plasma and Fletcher factor-deficient plasma. 4. It is concluded that inactive renin is activated by two separate proteolytic pathways: one pathway depends on both Hageman factor and plasma prekallikrein, and the other pathway depends on plasminogen. In the Hageman factor-dependent pathway plasma kallikrein and not Hageman factor is the major activator of inactive renin. It is assumed that pH 3·3-treatment of plasma destroys the major inhibitors of kallikrein and that pH 4·0-treatment destroys the major inhibitor of plasmin.


1989 ◽  
Vol 67 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Gregory M. T. Hare ◽  
Arlene Y. Loh ◽  
Daniel H. Osmond

Venous occlusion of the left arm in consenting men was induced for 10 or 20 min to stimulate local fibrinolytic and other proteases, thereby favouring the conversion of prorenin to renin. Using the two techniques cryoactivation and tryptic activation, we found that plasma active renin increased significantly after such occlusion (10 and 20 min) while prorenin rose more convincingly and progressively from 10 to 20 min. The renin increase can be partially attributed to hemoconcentration, but in vivo production and (or) local activation of prorenin to renin cannot be excluded. The prorenin rise can apparently be attributed to local extrarenal production, and not to hemoconcentration or influx, since it was progressive and neither prorenin nor renin levels were raised at all in blood circulating outside the occluded arm. Prekallikrein and plasminogen levels were elevated in occlusion plasmas, but responsibility of these enzyme systems for any enhanced activation of prorenin was not established. The trypsin inhibitory capacity was also elevated, increasing the requirement of trypsin to achieve optimal activation of prorenin, but not changing the prorenin estimate itself. Thus, prorenin appears to be released extrarenally, within the vasculature of an occluded arm, while in vitro evidence suggests that the mechanisms for its activation were stimulated. The importance of such extrarenal production and activation of prorenin for renin production under other physiological or pathophysiological conditions remains to be determined.Key words: venous occlusion, extrarenal prorenin, production, activation.


1987 ◽  
Vol 9 (8-9) ◽  
pp. 1383-1390 ◽  
Author(s):  
P. Dessì-fulgheri ◽  
F. Cocco ◽  
N. Glorioso ◽  
F. Bandiera ◽  
P. Madeddu ◽  
...  

Author(s):  
Hans-Peter Knopf ◽  
Frances Duffy ◽  
Ruben Papoian

We describe a time-resolved fluoroimmunoassay (TRFIA) for recombinant human interleukin-3 (IL-3). The assay design uses two different monoclonal anti-IL-3 antibodies, giving a two-site immunometric assay. The TRFIA for IL-3 is a direct adaptation from our existing ELISA for IL-3. By using the same assay configuration and exchanging a europium-streptavidin conjugate for alkaline phosphatase-streptavidin, we were able to minimize the time required to develop a TRFIA, but still increase the working range of the TRFIA over the ELISA by 20-fold. The limit of detection is 5 ng/L, with the limits of quantification set at 5 and 30 000 ng/L human plasma.


1987 ◽  
Vol 252 (4) ◽  
pp. E509-E518
Author(s):  
N. Glorioso ◽  
C. Troffa ◽  
J. H. Laragh ◽  
S. A. Atlas ◽  
D. Marion ◽  
...  

Inactive renin, prorenin, is found in high concentrations in human plasma. We report herein the characteristics of trypsin-activated inactive renin from cat kidney and plasma. Cat and human plasma inactive renin were activated by similar concentrations of trypsin. As in humans, there was more inactive than active renin in cat plasma; also, inactive renin was low but detectable after nephrectomy. Trypsin-activated renal inactive renin, purified on Cibacron blue agarose and pepstatin-amino-hexyl-Sepharose chromatography, was inhibited by pepstatin and by a renin inhibitor similarly to cat and human active renins. The pH optimum of cat renin was biphasic: the higher peak of active renin was at pH 5.7, whereas that of activated inactive renin was at pH 7.5. As in humans, active and inactive plasma renin increased during sodium depletion and inactive renin increased during beta-adrenergic blockade, while active renin decreased. These results demonstrate that cat inactive renin is similar to human prorenin. Therefore, the cat may be a useful model for the study of prorenin.


1987 ◽  
Vol 252 (1) ◽  
pp. F32-F38 ◽  
Author(s):  
D. Chansel ◽  
J. C. Dussaule ◽  
N. Ardaillou ◽  
R. Ardaillou

Renin activity was measured in the incubation medium, and the cellular extract of human mesangial cells, which had been cultured in the presence of renin-free human plasma (three kidneys; 4-7 passages). Active renin and total renin obtained after trypsin treatment was estimated by radioimmunoassay of angiotensin I using renin-free human plasma as a substrate. Mesangial cell renin had characteristics similar to those of standard human renin; optimum enzymatic activity at pH 5.8, marked inhibition in the presence of two (monoclonal and polyclonal) human renin-specific antibodies and of SR 42128, a new potent statine-containing renin inhibitory peptide. The synthetic capability of the mesangial cells varied markedly with the original kidney (1-49 and 0.3-0.9 ng X h-1 X mg-1 for total renin in the medium and the cellular extract respectively). Renin was secreted mainly as inactive renin. Prostaglandin E2 (PGE2) and carba-prostaglandin I2 (PGI2) (a stable analogue) produced a dose-dependent (0.1-1.10 microM) increase in renin activity in both the cellular extract and the culture medium. Isoproterenol (200 microM) increased renin activity only in the medium. The effects of these agonists were more marked on inactive than on active renin. These results demonstrate that cultured human mesangial cells synthesize and release renin in a stable manner over a long period of culture, thus providing a useful tool for the in vitro study of renin secretion and its control.


1980 ◽  
Vol 59 (s6) ◽  
pp. 29s-33s ◽  
Author(s):  
S. A. Atlas ◽  
J. H. Laragh ◽  
Jean E. Sealey ◽  
T. E. Hesson

1. Plasma prorenin (inactive renin), which accounts for about 70% of the total renin in human plasma, was almost completely separated from active renin by affinity chromatography on Cibacron blue F3G-A-agarose. The slight residual renin activity present in the prorenin peak can be removed on concanavalin A-Sepharose, demonstrating that prorenin is completely inactive. 2. The renin activity of both human renal cortical extract and renal perfusate increased after incubation with trypsin. This trypsin-activable renin accounted for 15 and 40% of the total renin in extract and perfusate respectively. 3. Trypsin-activable renin from both renal extract and renal perfusate was, like plasma prorenin, almost completely separated from active renin on Cibacron blue F3G-A-agarose. After additional chromatographic steps, the trypsin-activable renin from renal cortical extract was found to be completely inactive. 4. We conclude that human kidney contains, and is able to release, a trypsin-activable renin that resembles plasma prorenin. It may differ from many of the 60 000 molecular-weight forms of renin previously identified in renal extracts, since these possess considerable intrinsic renin activity and probably represent a complex of renin with a binding protein.


1988 ◽  
Vol 75 (6) ◽  
pp. 669-672 ◽  
Author(s):  
Jitsuo Higaki ◽  
Toshio Ogihara ◽  
Yuichi Kumahara

1. The plasma renin concentration (PRC) and active renin concentration determined by radioimmunometric assay (ARC) were measured before and after administration of frusemide or captopril to normal volunteers. 2. Injection of 40 mg of frusemide followed by 1 h in the upright position significantly increased both PRC (P < 0.001) and ARC (P < 0.001). Oral administration of 50 mg of captopril also increased PRC (P < 0.05) and ARC (P < 0.05). 3. ARC and PRC were linearly correlated in the basal supine position [y = 0.635 x − 0.048 (y = PRC, x = ARC), r = 0.958, P < 0.001], after frusemide injection and standing (y = 0.800x + 0.359, r = 0.793,P < 0.02) and after captopril administration in the supine position (y = 0.938x − 0.555, r = 0.998,P < 0.001). 4. A cross-calibration study in which pure human renin was added to pooled plasma and PRC and ARC were measured showed linearity between the values obtained by the two methods. 5. The regression line for values of PRC and ARC in the supine position after captopril administration had a significantly greater slope (P < 0.001) and that for values after frusemide injection and standing was significantly elevated (P < 0.001) compared with the regression line for basal values in the supine position. 6. These results show that the biological activity of renin may be increased by various acute stimulations of renin to inappropriately high levels compared with the immunological activity. This implies that the mode of processing of human plasma renin may be altered by acute stimulation of renin. Furthermore, current methods for assay of plasma active renin may give erroneous values in various pathophysiological conditions.


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