Two-site direct immunoassay specific for active renin.

A sensitive immunoradiometric assay, without an enzymatic step and specific for active human renin, was developed with use of two monoclonal antibodies (MAbs). In this assay system, the first MAb was coupled to magnetic beads (Magnogel); the second one, directed against the active form of the enzyme, was radiolabeled with 125I. The specificity of this assay was demonstrated in experiments measuring the active plasma renin concentration in the presence or absence of inactive renin. The assay, performed in two steps, was sensitive enough to detect 0.9 pg of renin per tube (3.5 ng/L). Intra- or interassay CVs were < 10%. Concentrations of active plasma renin measured in normotensive subjects were between 7 and 40 ng/L.

Investigation of the biochemical effects of renin inhibition in normal volunteers treated by an ACE inhibitor.

1. In order to investigate accurately the biochemical effects of renin inhibition in man, we have developed a sensitive assay to measure angiotensin I (1-10) decapeptide. 2. Angiotensins were extracted from plasma by adsorption to phenylsilylsilica, and angiotensin I (Ang I) was quantified by radioimmunoassay. The detection limit was 0.77 fmol ml-1, and the extraction recovery of [125I]-Ang I added to albumin buffer was 83% at the inflection point (10 fmol ml-1) of the standard curve. The overall recovery was 98.5 +/- 3.5%. The intra- and inter-assay reproducibility was 10.4% and 9.7% respectively. Cross-reactivity of the antiserum used was low (less than 0.3%) with all angiotensin peptides tested except Ang (2-10) nonapeptide. 3. A human pharmacological model was subsequently used to assess in vivo the biochemical effects of the renin inhibitor CGP 38560A. Six healthy volunteers received 20 mg lisinopril, a long-acting ACE-inhibitor. During the following 24 h, the renin-angiotensin system was reset with typically elevated active plasma renin and Ang I, at respectively 275 and 429% of basal values. 4. In a randomized three-way cross-over protocol, the six volunteers received a 30 min infusion of the renin inhibitor CGP 38560A (125 or 250 micrograms kg-1) or 5% glucose. The fall in plasma Ang I was 92% and 97.5% after the lowest and highest dose of the renin inhibitor, respectively. A concomitant increase in active plasma renin was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

[Recent advances in the investigation of the renin-angiotensin and cardiac natriuretic systems in patients with chronic heart insufficiency]. / Progrès récents dans l'exploration du système rénine-angiotensine et du système cardiaque natriurétique chez des patients en insuffisance cardiaque chronique.

The renin-angiotensin and cardiac natriuretic systems were studied by measuring plasma renin activity, plasma concentrations of active renin, angiotensinogen, atrial natriuretic hormone and urinary cyclic GMP in 37 patients with moderate to severe cardiac failure. The plasma sodium and osmolality were chosen as markers of hydroelectrolytic imbalance and plasma concentrations of préalbumin and retinol-binding protein as indicators of the degree of hepatocellular dysfunction. Plasma renin activity (PRA) plasma concentration of active renin, atrial natriuretic hormone and urinary cyclic GMP were higher in patients in NYHA Class IV than in those in Classes II-III, whilst plasma sodium, angiotensinogen, prealbumin and retinol-binding protein concentrations were lower in Class IV patients than in patients in Classes II-III. The plasma angiotensinogen concentrations were negatively correlated with PRA (r = -0.41, p less than 0.02), active renin (r = -0.45, p = 0.005), the atrial natriuretic factor (r = -0.36, p less than 0.05) and positively correlated with prealbumin (r = 0.54, p less than 0.001) and retinol-binding protein (r = 0.60, p less than 0.0001). In NYHA Class IV patients the decreased circulating renin substrate led to an underestimation of plasma concentrations of active renin by measurement of PRA. On the other hand, direct radio-immunometric measurement of active renin allows true estimation of circulating active renin, independently of plasma angiotensinogen concentrations and thereby reliably reflects activation of the renin system. The serum sodium was negatively correlated with active renin (r = -0.66, p less than 0.0001) in these patients not receiving converting enzyme inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effects of deprivation and replacement by percutaneous 17 beta estradiol and oral progesterone on blood pressure and metabolic parameters in menopause patients with non-insulin-dependent diabetes]. / Effets de la substitution par 17 beta estradiol percutané et progestérone orale sur la pression artérielle et les paramètres métaboliques chez des patientes ménopausées diabétiques non insulinodépendantes.

The main purpose of this randomized controlled study was to assess the effects of postmenopausal estrogen replacement therapy on blood pressure (BP) and plasma renin substrate (PRS) in non insulin-dependent diabetic patients (DNID). We randomized 32 postmenopausal DNID (mean age: 55.3 +/- 4.2 years) into two groups: 16 women were untreated, and 16 received percutaneous estradiol (E2) 17 beta and natural progesterone for 6 months. Systolic (SBP) and diastolic (DBP) blood pressure were monitored by an automatic device at inclusion and on the 1st, 3rd and 6th months of therapy. Treatment efficacy was proven by significant E2 plasma increase to 92.2 +/- 13.4 pg/ml in the treated group, which is a sufficient level for preventing postmenopausal osteoporosis. No significant inter or or intra-individual variation in SBP or DBP was observed in either group. The same stability was noted for plasma renin substrate. No significant difference was noted between the two groups in terms of body weight, fructosamine and glycosylated hemoglobin A1c after 1, 3 and 6 months. There was also no change in plasma levels of total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides and apolipoproteins A1 and B. All the patients who received replacement therapy wished to continue treatment. We conclude that the association of percutaneous E2 17 beta and natural progesterone had no deleterious effects, in diabetic patients, on BP, carbohydrate and lipoprotein metabolism. Thus this postmenopausal replacement therapy appears preferable in this vascular high risk population, particularly since estrogens via the parenteral route may have an antiatherogenic effect by direct action on the vessel walls.

[Use of intravenous nicardipine for the diagnosis of renovascular hypertension]. / Utilisation de la nicardipine par voie intraveineuse pour le diagnostic d'hypertension rénovasculaire.

UNLABELLED: The diagnosis of remediable renovascular hypertension (RVH) requires demonstration of lateralization of renal vein renin (RVR). In order to increase the accuracy of RVR ratio, we investigated the acute effects of a bolus of Nicardipine (Nic.: 4 mg i.v.) on hemodynamics and RVR in 19 patients. 13 patients had an unilateral renal artery stenosis greater than 75% (RVH: 10 atheroma and 3 fibrodysplasia) and 6 patients had essential hypertension (EH). In 6 patients (5 RVH and 1 EH) treatment could not be discontinued and only a monotherapy by central alpha-agonists was prescribed. RVR samples were obtained 15 minutes after renal vein catheterization (baseline values: T0) and 10 minutes after Nic. injection (T10). During the whole procedure, mean blood pressure (MBP) and heart rate (HR) were monitored every 2 minutes by an automatic device. Active renin was measured by a new immunoradiometric assay. A RVR ratio (stenotic/contralateral side) greater than 1.5 was considered as a positive ischemic index. RESULTS: The relative changes in MBP and HR between T0 and T10 were of same magnitude in both groups. No patient suffered any untoward effect from the fall in MBP. Nic. increased RVR release from both sides in RVH group as well as in the EH group. At baseline, 6/13 of the RVH patients and none of the EH patients had a RVR greater than 1.5. After Nic. injection, all the RVH patients had a RVR greater than 1.5 and none among EH patients. We conclude that single i.v. Nic. bolus is a safe and a reliable procedure which increases diagnostic accuracy by enhancing RVR when there is an unilateral RVH disease.

Low angiotensinogen levels are related to the severity and liver dysfunction of congestive heart failure: implications for renin measurements.

PURPOSE: To compare the determination of plasma renin activity (PRA) and the direct measurement of active renin by immunoradiometric assay (IRMA) as methods of assessing the renin system in patients with congestive heart failure. PATIENTS AND METHODS: The status of the renin-angiotensin system in congestive heart failure was assessed by measuring the plasma renin substrate concentration, PRA, and plasma concentration of active renin in 37 patients with mild to severe congestive heart failure. Natremia and plasma levels of atrial natriuretic factor (ANF) were determined as biologic indexes of the severity of heart failure, and concentrations of prealbumin and retinol-binding protein were used as indexes of liver dysfunction. RESULTS: The PRA and the concentrations of active renin and ANF were markedly higher in patients with New York Heart Association class IV heart failure than in patients with class II to III heart failure, while natremia and the concentrations of renin substrate, prealbumin, and retinol-binding protein were markedly lower in the class IV patients than in the class II to III patients. Plasma renin substrate concentration was negatively correlated with active renin concentration (n = 37, r = -0.45, p = 0.005), and positively related to natremia (r = 0.56, p less than 0.0005), prealbumin (r = 0.54, p less than 0.001), and retinol-binding protein (r = 0.60, p less than 0.0001). CONCLUSIONS: Low levels of plasma renin substrate can be considered as an indirect index of the severity of heart failure that reflects both the high level of circulating active renin and the decrease in hepatic protein output. In patients with class IV heart failure, low levels of renin substrate led to a marked underestimation of active renin concentration from measurements of PRA. In contrast, direct IRMA of active renin measures the true plasma active renin concentration, independent of plasma renin substrate, and closely reflects renin secretion.

Pharmacological investigations of a new renin inhibitor in normal sodium-unrestricted volunteers.

1. CGP 38 560 A, a low-molecular-weight, non-peptidic renin inhibitor, was well tolerated upon intravenous and oral administration to recumbent healthy volunteers on an unrestricted-sodium diet. 2. After intravenous infusion over 30 min at a rate of 100 ml h-1, doses of 50, 125 and 250 micrograms kg-1 appear to induce a long-lasting inhibition of plasma renin activity. Plasma angiotensin II was decreased in a dose-dependent manner during the infusion and thereafter reverted to the initial level. A concomitant dose-related increase in active plasma renin was observed. Blood pressure was unaffected. The plasma levels of CGP 38 560 reached during infusion were at least 2000-fold higher than the theoretical inhibitory concentration based on in vitro results. 3. After oral administration in doses of 50, 100 and 200 mg CGP 38 560 A, inhibition of plasma renin activity was observed, but plasma active renin was unchanged. Blood pressure also remained unaffected. 4. CGP 38 560 was rapidly cleared from plasma with a half-life of 7.6 min for the first phase and 63 min for the second phase. Plasma levels were 100-fold lower after oral administration than after infusion, indicating a low degree of absorption (less than 1% oral bioavailability).

Epithelioid sarcoma of soft tissues: a case of extrarenal renin-secreting tumour.

The case of a young woman presenting with a renin-secreting soft tissue sarcoma is described. The primary extrarenal tumour as well as metastatic disease were associated with severe hypertension and both required surgical treatment. The location of these rare malignant tumours and their association with renin-dependent hypertension is discussed. In cases of this type, reappearance of hypertension suggests tumour recurrence.

Converting enzyme inhibition and kidney function in normotensive diabetic patients with persistent microalbuminuria.

The effects of a long term reduction in blood pressure on the kidney function of normotensive diabetic patients who had persistent microalbuminuria (30-300 mg albumin/24 hours) were studied in two groups of 10 such patients before and during six months of treatment with either 20 mg enalapril or placebo daily. Treatments were assigned randomly in a double blind fashion. Before treatment both groups had similar clinical characteristics, weight, diet, total glycosylated haemoglobin, median albumin excretion rate (enalapril group 124 mg/24 h, placebo group 81 mg/24 h), and mean arterial pressure (enalapril group 100 (SD 8) mm Hg, placebo group 99 (6) mm Hg). During treatment weight, urinary urea excretion, and total glycosylated haemoglobin remained unchanged. The mean arterial pressure decreased in the enalapril group but not in the placebo group (enalapril group 90 (10) mm Hg, placebo group 98 (8) mm Hg). The median albumin excretion rate also fell in the enalapril group but not in the placebo group (enalapril group 37 mg/24 h, placebo group 183 mg/24 h.) The glomerular filtration rate rose in the enalapril group from 130 (23) ml/min/1.73 m2 to 141 (24) ml/min/1.73 m2, and total renal resistances and fractional albumin clearance decreased while fractional albumin clearance increased in the placebo group. These results show that in patients who have diabetes but not hypertension a reduction in blood pressure by inhibition of converting enzyme for six months can reduce persistent microalbuminuria, perhaps by decreasing the intraglomerular pressure.

Rapid measurement of total and active renin: plasma concentrations during acute and sustained converting enzyme inhibition with CGS 14824A.

Total and active renin were measured in plasma of 6 normal volunteers before and after acute and sustained angiotensin converting enzyme (ACE) inhibition with CGS 14824A (2 mg and 10 mg p.o. q.d.) or placebo treatment. The same sandwich technique was used for the measurement of both total and active renin using a polyacrylamide-iron-oxide linked monoclonal antibody to trap renin and 125I-labelled second monoclonal antirenin antibodies without or with specificity for active renin. Normal values for supine subjects ranged for active renin between less than 3 pg/ml and 28 pg/ml and for total renin between 73 and 263 pg/ml. Plasma ACE activity was clearly suppressed during 24 hours following both 2 mg and 10 mg CGS 14824A. Active plasma renin reached 6- and 12-fold normal values on days 1 and 7 of treatment with the 10 mg dose. Total renin rose to 150% and 228% respectively at the same time. Inactive renin continued rising during the first day of 10 mg CGS treatment to a final 141% at 24 hours post-drug and didn't change on day 7. Plasma renin activity correlated well with active renin levels (r = 0.92). We conclude that both total and active plasma renin concentrations can now be directly measured with great accuracy within 6 hours.

[Progestogen contraception using chlormadinone acetate in women presenting high vascular risk. (A gynecoendocrine, metabolic and vascular study)].

PIP: 20 women with metabolic, vascular, or gynecoendocrinological contraindications to use of combined oral contraceptives (OCs) or norsteroid progestins participated in a 6-month study of chlormadinone acetate, a progestin derived from 17-OH progesterone known for its weak androgenic activity. 5 mg doses of chlormadinone acetate were administered on the morning and evening of the 6th to the 26th cycle day for 3 woman and on the 8th to the 26th cycle day for 17 others. Blood tests were conducted during the luteal phase after fasting. The average age of the study subjects was 29.9, the average weight was 57.8 kg, and the average height was 161 cm. There were no significant variations over the course of the study in weight, blood pressure, renin substrate, high density lipoprotein (HDL) cholesterol, triglycerides, plasma apo-B, or antithrombin III. There was a significant reduction in apoprotein A1, the principle protein fraction of high density lipoproteins, from the 3rd to the 6th cycles, and a nonsignificant reduction of the apoprotein B. The ratio of A1/B apoproteins was not significantly modified. There were no signs of hyperestrogenism, hyperandrogenic effects, or digestive intolerance. In most cases there was no significant change in cycle duration or intensity of menstrual bleeding. 3 patients were recurrently amenorrheic. The amount of bleeding was considered normal by 14 patients and diminished by 3. There were no cases of menorrhagia and 5 of minor metrorrhagia in the first 3 months of use. No pregnancies occurred. After voluntary termination of contraception, 2 patients rapidly became pregnant. Measurement of ovarian hormone levels and gonadotropins indicated that chlormadinone acetate at the prescribed dose completely inhibited progesterone secretion in all patients and considerably reduced the production of estradiol in the luteal phase. Chlormadinone acetate has the dual advantages of avoiding the estrogen-induced side effects of combined OCs and avoiding hyperestrogenism. The use of a progestin derived from 17-OH progesterone may offer a contraceptive method suitable for women with metabolic or vascular contraindications to combined OCs or gynecoendocrinological contraindications to low dose progestins, who are unable or unwilling to use mechanical or local contraceptives. Chlormadinone acetate should however remain a method for use under exceptional circumstances.^ieng

Direct immunometric assay of active renin in human plasma.

None of the methods currently available can detect the small numbers of active renin (AR) molecules present in plasma. Among seven monoclonal antibodies (Ab), two Abs were selected which did not recognize the same epitope and could be used in a sandwich assay. The first monoclonal Ab, 3E8, binds soluble renin (B 50% = 1 x 10(-10) mol/l) and does not inhibit its enzymatic activity. It was coupled to magnetic beads (Magnogel) and was used to trap both active and inactive renin from 250 microliters plasma. The second Ab, 4G1, binds renin (B 50% = 3.5 x 10(-10) mol/l), inhibits its enzymatic activity, and recognizes inactive renin less than AR. It was iodinated and used to detect AR trapped on Magnogel by the first Ab during a 4-h incubation. The assay can detect 16 pg/ml in human plasma and is highly reproducible. The AR level of 15 normotensive subjects, aged 20-45 years, in an upright posture and on a normal sodium intake, was found to be 41 +/- 18 pg/ml (MRC renin standard). The plasmas were trypsin-activated and their total renin levels were measured with the same pair of monoclonal Abs. The mean value of 286 +/- 142 pg/ml is similar to the value obtained by other assay systems which measure total renin with Abs recognizing both active and inactive renin. The direct measurement of AR provides a convenient and standardized method, since the production of the two monoclonal Abs is unlimited.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnosis and treatment of renin-secreting tumors. Report of three cases.

During the past 10 years, we have found renin-secreting renal juxtaglomerular cell tumors in three hypertensive patients (two women, one man, aged 22, 69, and 21 years, respectively). The major chemical and biological findings revealed the association of severe hypertension with hypokalemia and increased plasma renin activity and plasma aldosterone. The diagnosis of such tumors is difficult, and two of the three patients were followed up for four and five years respectively before undergoing surgery. The pharmacological blockade of the renin system by various agents (beta-blockers, angiotensin II antagonists, and captopril) and its effects on blood pressure and plasma renin activity proved to be unreliable. Renal venous catheterization for renin measurements failed to provide adequate localization of the tumor. Direct radioimmunoassay, however, showed the total plasma renin to be markedly elevated. In addition, renal arteriography showed an avascular area corresponding to the renin-secreting tumor in each of the three patients. All three patients were cured of hypertension and hypokalemia by excision of the tumor.

Enzymatic activity of renin in plasma of normal and uraemic subjects.

Plasma renin reactivity (PRR) is the rate of angiotensin I production after addition of renin to plasma, minus endogenous renin activity. PRR is increased in plasma of patients with renal failure compared with that of normal subjects. The present study was carried out to determine if increased PRR in uraemic plasma is related to differences of endogenous active or inactive renin, endogenous renin substrate, or pH of the incubation in vitro. PRR in plasma of ten uraemic patients was greater (P less than 0.02) than that in plasma of ten normal subjects in incubations carried out at pH 7.4 and 5.7. Increased PRR was not accounted for by differences of endogenous active and inactive renin activity. After addition of renin, renin concentration (measured by direct radioimmunoassay) did not differ in normal and uraemic plasma. Renin substrate concentration, measured both indirectly and by direct radioimmunoassay, also did not differ in normal and uraemic plasma. Increased PRR in uraemic plasma is not related to alterations of renin or renin substrate concentrations. These observations are consistent with our earlier hypothesis that there is a deficiency of a renin inhibitor in uraemic plasma.

Improved diagnosis of unilateral renal artery lesions after captopril administration.

Captopril was administered (1 mg/kg of body weight) to 37 unselected hypertensive patients undergoing bilateral renal vein catheterization to determine its safety and efficacy in diagnosing hypertension related to unilateral renal artery lesions. In the 18 patients who had a unilateral renal artery lesion demonstrated by angiography, the ratio of plasma renin activity of the involved to uninvolved renal vein rose significantly after administration of captopril, whether or not patients were taking antihypertensive medication. This postcaptopril ratio differentiated cases of unilateral lesions from cases of bilateral lesions or absence of lesions without any overlap. The test was well tolerated despite occasional large drops in blood pressure. These data show that converting enzyme inhibition increases the diagnostic accuracy of renal vein catheterization by increasing the difference between the amount of plasma renin secreted by the two kidneys in cases of unilateral renal artery lesions.

Direct radioimmunoassays of renin and renin substrate during converting-enzyme inhibition.

In 10 severely hypertensive patients, on a low sodium diet, converting enzyme inhibition increased plasma renin activity and decreased plasma renin substrate. The use of direct radioimmunoassays for both the enzyme and its substrate showed that the number of immunoreactive renin molecules increased from 11.3 +/- 4.9 to 31.7 +/- 25.3 pmol 1(-1) whereas the number of immunoreactive renin substrate molecules decreased from 1.04 +/- 0.35 to 0.74 +/- 0.16 mumol 1(-1). The direct radioimmunoassay for angiotensinogen gave higher values than the direct enzymatic assay, and during converting enzyme inhibition, the difference between both methods increased in proportion to the rise in circulating renin. It is concluded that the difference between the renin substrate radioimmunoassay, which measures angiotensinogen and des-angio I-angiotensinogen, and the renin substrate enzymatic assay which only measures "active" substrate, is an index of the increased consumption of renin substrate, in a situation where the fall in angiotensin II enhances renin release and decreases renin substrate release.

Direct radioimmunoassay of human renin.

Antibodies were raised in rabbit against pure human renin. The antisera obtained are highly specific for human renin versus hog, dog and rat renin. They do not cross-react with acid proteases such as pepsin and human cathepsin D. A direct radioimunoassay is described for human renin in plasma and kidney extracts. 30 to 50 pg of enzyme (2.5 to 4 x 10(-5) Goldblatt units) are detected.