Biochemical properties of renin in human pituitary tissue

The biochemical properties of renin, extracted from human pituitary specimens obtained at autopsy, were studied using a specific antirenin antibody raised against human kidney renin. The following results were obtained. The molecular weight of pituitary renin was estimated to be about 37,000 daltons by gel filtration through Sephadex G-100. The optimum pH of pituitary renin was between 6.0 approximately 7.0, while that of a renin-like substance which did not react with the antirenin antibody had an acidic pH of 4.0, with a pH comparable to that of the cathepsin D-like enzyme in the pituitary tissue. The presence of two different isoelectric-point species of pituitary renin was revealed by isoelectric focusing, one with a point of pH 4.47 and the other with that of pH 5.77. The Km value of pituitary renin was 37.9 microM for synthetic human renin substrate. Affinity chromatography of the pituitary renin on a Concanavalin-Sepharose column showed that most (87.4%) of the pituitary renin did not contain glycoprotein residues. Treatment with either trypsin or glandular kallikrein increased the renin activity, indicating the presence of an inactive form of renin in the pituitary tissue. From these findings, it is concluded that specific renin exists in human pituitary tissue. It seems likely that the pituitary renin is of local origin rather than contamination of the circulating enzyme.     

Occurrence of big renin in human plasma, amniotic fluid and kidney extracts.

Big renin, a relatively inactive renin which possesses a molecular weight larger than that of normal plasma or renal renin, has been demonstrated by gel filtration in certain human plasma, tumor extracts, and amniotic fluid. Big renin was not present in normal plasma or kidney extracts. Plasma from 3 hypertensive patients with nephropathy contained chiefly big renin. Varying proportions of both big and normal renin activity were present in plasma of other patients with hypertension and proteinuria. The renin present in amniotic fluid, which increased in activity following exposure to acid pH, was shown to be big renin in two patients. Large amounts of circulating big renin apparently can cause hypertension in patients with Wilms' tumors. Furthermore, the relatively inactive big renin may replace normal plasma renin in some patients, resulting in low plasma renin activity.     

Modification of renin reactivity by lipids extracted from normal, hypertensive, and uremic plasma.

Plasma renin reactivity (PRR), the in vitro rate of angiotensin generation after addition of renin, is greater in plasma of hypertensive patients and uremic patients than in plasma of normotensive control subjects. To determine if this difference is due to different substrate reactivities, substrate was denatured and replaced with homologous substrate. After a 180 min incubation, PRR in normal plasma (73 ng/ml +/- 5 SE) was less (P less than 0.01) than that in hypertensive (112 ng/ml +/- 15 SE) or uremic (123 ng/ml +/- 39 SE) plasma. To determine if uremic plasma lacks a renin inhibitor, buffer or plasma was added to renin-renin substrate. Less angiotensin was generated (P less than 0.05) with normal (72 ng/ml +/- 4 SE) and uremic (88 ng/ml +/- 4 SE) plasma during 30 min than with buffer (107 ng/ml +/- 4 SE). After 180 minutes, less angiotensin was generated with normal (P less than 0.05) but not uremic plasma (P greater then 0.6), than with buffer. In vitro angiotensin generation was inhibited by lipids extracted from normal plasma. Lipids were separated into acetone soluble (neutral lipids) and acetone insoluble (phospholipid) fractions. Acetone soluble lipids, extracted from normal plasma, competitively inhibit renin: renin was not inhibited by acetone insoluble lipids. Acetone soluble lipids extracted from uremic plasma inhibited PRR to a lesser extent than lipids from either normal plasma or hypertensive plasma (P less than 0.01). Increased PRR in uremic plasma may be related to the deficiency of a circulating acetone soluble renin inhibiting factor.     

Cryoactivation of inactive renin in human plasma.

The mechanism of the increase in renin activity in human plasma which had been kept -5 degrees C for 4 days (cryoactivation) was investigated. From the results of clinical studies, it is likely that the controling mechanism of inactive renin has something in common with that of active renin. The experimental data showed that the increase in renin activity of human plasma by cryoactivation was closely correlated to the increase obtained by incubation with trypsin (r = 0.88, p less than 0.001, n = 10). Soybean trypsin inhibitor, aprotinin and di-isopropylfluorophosphate (DFP) inhibited cryoactivation, indicating that the cryoactivation is due to the action of a trypsin-like serine enzyme. Trypsin which had no effect on plasma renin activity in the presence of the same amount of soybean trypsin inhibitor at 37 degrees C, activated the renin activity during cold incubation, suggesting that the dissociation of the trypsin-inhibitor complex may have taken place at a low temperature. Endogenous trypsin inhibitor is also likely to lose its affinity to endogenous trypsin-like enzyme at a low temperature.     

A comparison of cold and acid activation of big renin and of inactive renin in normal plasma.

Normal human plasma contains "inactive renin," whose ability to generate angiotensin I increases after exposure to pH 3.3. Big renin is a partially inactive enzyme of larger molecular weight, which is also activated at pH 3.3, and is found of pregnant women, and in amniotic fluid, but not in normal plasma. We have compared the effects of acid exposure and storage at 4 and -4 C on normal plasma and plasma containing big renin. The concentration of inactive renin in normal plasma was approximately equal to that of normal active renin, and its activity increased slowly on prolonged standing at -4 but not 4 C. In contrast, the activity of big renin increased by 50% as early as 1-3 days at 4 C and increased even more quickly at -4 C. Acid treatment of plasma containing big renin caused 4-10 times greater increase in active renin than similar treatment of normal plasma. During gel filtration, both cold-activated and previously acidified big renin coeluted with unactivated big renin. These data indicate that big renin is highly susceptible to cold or acid activation and that such activation of big renin does not result in a detectable decrease in its molecular weight of 60,000 daltons. Furthermore, acid and cold seem to activate the same pool of inactive renin in normal plasma. Although both normal and big renin are stable for long periods below -20 C, a serious overestimate of plasma renin activity can occur if plasma is stored just above its freezing point before assay.     

Inhibition of human plasma renin activity by pepstatin.

Pepstatin, a pentapeptide isolated from streptomyces, is a powerful inhibitor of several acid proteases. Its ability to inhibit the renin-angiotensinogen reaction was studied in vitro, in various human plasma. A 50% inhibition of plasma renin activity was obtained with 10(-6)M pepstatin at pH 5.7 and 10(-5)M at pH 7.4. The inhibition of plasma renin activity by pepstatin was studied in hypertensive patients with various plasma renin levels. The inhibitory effect could be demonstrated in patients with low, normal and high renin activity at both pH's. The type of inhibition and the inhibitory constant were investigated by Dixon plot and Lineweaver-Burk plot in three separate experiments. On both representations, a competitive type of inhibition was found with an inhibitory constant of about 1.2 x 10(-6)M.     

Measurement of plasma renin concentration using exogenous human renin substrate in normal subjects: correlation with plasma renin concentration and plasma aldosterone concentration.

Measurement of plasma renin concentration (PRC) was done in normal subjects at rest and under acute stimulation of renin release under unrestricted sodium intake. Concurrent measurements of plasma renin activity (PRA) and plasma aldosterone concentration (PA) were carried out. The mean values of PRC at rest and after stimulation of renin release were 12.8 +/- 1.3 (SEM) and 21.7 +/- 4.4 (SEM) ng AT I/ml/h, respectively. These corresponded to renin contents of 3.4 +/- 0.34 (SEM) X 10(-5) Goldblatt units and 5.8 +/- 0.36 (SEM) respectively. The mean percent increase of PRC (82.1 +/- 19.3 (SEM)) %) was almost indentical to that of PA (81.5 +/- 16.4 (SEM) %), but differed from that of PRA (269 +/- 83.1 (SEM) %). A very high correlation between concurrent PRC and PA (r = 0.92, P less than 0.001) was found in normal subjects at rest and under acute stimulation of renin release. A good correlation between PRC and PRA (r = 0.85, P less than 0.001) was also observed. However, a higher correlation between percent increases of PRC and PA (r = 0.92, P less than 0.001) than that of PRA and PA (r = 0.80, 0.01 less than P less than 0.005) was found. Results show that PRA is a good index of the renin content in plasma in normal subjects at rest and PRC reflects actual renin concentration in plasma at rest as well as under stimulation of renin release.     

Binding of human renin to plasma proteins and comparison with plasma active and inactive renin

Inactive renin was partially purified from normal male plasma. It showed no enzymatic activity at pH 7.4, and its molecular weight by gel filtration was 53 000 compared with 45 000 for partially purified plasma active renin and 41 000 for pure renal renin. After exposure to pH 3.0 the inactive renin became enzymatically active but its molecular weight did not change. The acid-activation could be reversed when the renin was re-adjusted to pH 7.4 and warmed. Pure renal renin labelled with I125 was added to normal male plasma. It had a molecular weight of 40 000 by gel filtration. When the mixture was acidified and neutralized, some I125 label appeared in a high-molecular-weight peak, as might occur if the renin was associated with a binding protein. If the mixture of plasma and labelled renin was treated with guanidine hydrochloride, most of the label appeared in the high-molecular-weight peak. However, after both acid treatment and treatment with guanidine hydrochloride the 'high-molecular-weight' peak appeared in the void volume of the column (Mr 108 000) rather than in the position of inactive renin (Mr 53 000). Also, the I125 label in the peak was neither immunologically nor electrophoretically similar to renin. It may represent denatured renin bound to plasma proteins, e.g. alpha-2 macroglobulins. We conclude that 'inactive renin' is a prorenin-like material rather than a protein-bound form of active renin.     

Normal plasma renin activity in low renin hypertension.

Plasma renin activity (PRA) was measured every 4 h during a 24-h period of continuous recumbency in 10 patients with essential hypertension. All had maximum values at 12 midnight, 4 AM or 8 AM. Analysis of our date and that of others indicates that in some patients with "low renin" hypertension (LRH) these noctural peaks are of normal magnitude, occasionally exceeding values obtained after four hours of erect posture. Sleep-induced renin release in these patients is not suppressed despite blunted responses to other stimuli. This suggests that the ability to synthesize and release renin may be normal in these patients. Such peaks were not obserived in all LRH subjects, however. The area enclosed by the recumbent PRA curve was also normal in some, but not all patients with LRH. Low renin hypertension may not be a homogeneous disorder.     

Effects of aging on human plasma renin: simultaneous multiple assays of enzyme activity and immunoactivity of plasma renin

The effects of aging on plasma renin in normotensive volunteers were evaluated by conventional indirect RIA of angiotensin I and a newly developed direct RIA. Plasma renin activity and the plasma concentration of active renin measured by radiometric assay with monoclonal antibody were significantly lower in 14 subjects over 60 years than in 15 subjects under 60 years (plasma renin activity: 0.5 +/- 0.1 vs 1.7 +/- 0.4 nmol.1-1.h-1, P less than 0.01; plasma active renin: 0.50 +/- 0.05 vs 0.87 +/- 0.13 pmol/l, P less than 0.01, means +/- SEM), whereas neither the total renin activity nor the total plasma renin concentration measured by the newly developed immunometric assay were different in the two groups. In another study, the plasma renin concentration, total renin concentration and immunoreactive total renin concentration measured by direct RIA with polyclonal antibody were determined in 17 young (less than 60 years) and 12 elderly (greater than or equal to 60 years) subjects. Plasma renin concentration was significantly lower in the elderly subjects (1.7 +/- 0.2 nmol.l-1.h-1) than in young subjects (3.2 +/- 0.7 nmol.l-1.h-3, P less than 0.05), but the total renin concentration and immunoreactive total renin concentrations in the two groups were not significantly different. These results indicate that the total renin content of the plasma does not change, whereas the active renin content decreases with age in normal subjects, and suggest that activation of prorenin to active renin may be impaired in elderly subjects.     

Measurement of plasma total renin by the anti-human renin monoclonal antibodies.

The authors evaluated the assay performances and clinical usefulness of a newly developed solid phase radioimmunoassay (RIA) for total renin concentration (TRC) in human plasma. The direct total renin RIA was performed by a sandwich technique with a pair of anti-human renin monoclonal antibodies. Renin activation with trypsin did not change TRC. The RIA showed satisfactory assay performances and demonstrated full compatibility with a direct RIA-kit for active renin concentration (ARC) in human plasma. The values of TRC were 105.3 +/- 8.6 pg/mL in normal subjects and 136.5 +/- 14.6 pg/mL in patients with essential hypertension. The values of TRC and the ratios of ARC to TRC were high in patients with renovascular hypertension and were low in patients with primary aldosteronism. Although the TRC value in diabetic patients was 134.4 +/- 14.8 pg/mL, the ratio of ARC to TRC was low. The RIA procedure was simple since prior purification or activation of renin was not required. These results suggest that the total renin RIA and its combined use with the active renin RIA may be helpful in understanding the renin-angiotensin system in human plasma.     

A large form of renin from normal human kidney.

The apparent molecular size of circulating human plasma renin is 43,000 daltons. In this report, the Stokes' radius of renin extracted from human kidney cortex in low ionic strength buffer in the presence of protease inhibitors was shown to correspond to an apparent molecular weight of 58,000 +/- 3,000. If protease inhibitors are omitted, if the kidney tissue is frozen and thawed multiple times, or if the kidney extract is acidified to pH 2.8, renin activity of an apparent molecular size of 42,000 +/- 3,000 can be identified. Both species of renin bind to an affinity support bearing the substrate analog inhibitor His-Pro-Phe-His-Leu-D-Leu-Val-Tyr. Antibody raised to the higher molecular form of the enzyme inhibits the activity of both forms in an equivalent manner. These observations suggest that the larger form of renin may be a biosynthetic precursor of plasma renin, either in the form of a zymogen or an enzyme-binding protein complex.     

Direct radioimmunoassay of human renin: comparison with renin activity in plasma and amniotic fluid

Human plasma and amniotic liquid were activated by dialysis at pH 3.3. Then, renin before and after acidification was determined by two methods: enzymatic activity measurement, and direct radioimmunoassay. The identity between nonactivated and activated renin in plasma and amniotic fluid on the one hand, and pure renin on the other, was demonstrated by the dilution curves in radioimmunoassay. After acidification, mean plasma renin activity in 17 patients with high renin activity rose from 26.8 +/- 11.7 pmoles A I ml-1 h-1 to 67.9 +/- 29.3 pmoles A I ml-1 h-1, whereas the mean renin concentration tested by direct radioimmunoassay remained constant at 13.8 +/- 10.5 and 14.8 +/- 11.2 fmol/ml before and after acidification respectively. In amniotic fluid, renin activity increased from 9.7 to 227 pmoles angiotensin I/ml/h, but the renin concentration did not change. Direct radioimmunoassay of renin may therefore be considered as measuring total renin, regardless of its enzymatic activity. In 12 hypertensive patients undergoing bilateral renal-vein catheterization, the direct measurement of renin was very significantly correlated to the non-activated (r = 0.883) and activated renin values (r = 0.963).     

Sandwich radioimmunoassay of human plasma renin using two monoclonal antibodies

Plasma immunoreactive renin was measured by sandwich radioimmunoassay, under various physiological and pathological conditions. Enzymatic activities of active renin and trypsin-activatable inactive renin were also measured. There was a significant correlation between plasma immunoreactive renin concentration (IRRC) and total (active plus inactive) renin concentration, as estimated by enzymatic activity. In plasma from normotensive volunteers and hypertensive patients, the IRRC were 279 +/- 37 pg/ml and 268 +/- 29 pg/ml, respectively. After the intravenous injection of furosemide, the plasma IRRC in normotensive volunteers increased significantly. IRRC was significantly higher in plasma from juvenile diabetics than in plasma from age-matched disease-free children. Thus, renin secretion in children with diabetes mellitus is increased.     

Electrophoretic characterization of active renin from human kidney and inactive renin from a human chorionic cell culture

Enzymatically inactive human renin from chorionic cells in culture is significantly distinct in polyacrylamide gel electrophoresis (pH 8.17, 0°C) from active human kidney renin. The inactive renin is larger and more basic than the active renin; their molecular weights derived from gel electrophoretic retardation coefficients relate as $\text{47.5}\text{35.3}$ kDa, their valences (net protons/molecule) as $\text{2.14}\text{1.85}$. In gel electrofocusing conducted in a mixture of simple buffers, both inactive and active renins exhibit 2 components at the steady-state. The molecular size and basicity of inactive renin are consistent with the hypothesis that it may be a precursor (prorenin), although the possibility that it is an inhibitor complex cannot be ruled out.     

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.