Plasma Inactive Renin Research Articles

Similarity between active and trypsin-activated inactive renin in dog plasma by means of renin inhibition: the dog as an animal model for studies of inactive renin.

A method for trypsin-activation of dog plasma inactive renin is described. Liquid phase trypsin (final concentration 6.7 mg/ml) was used and the reaction was stopped after 2 min at 4 degrees C by soybean trypsin inhibitor (13 mg/ml). Renin was measured as angiotensin I (Ang I) generation in trypsin-treated and untreated plasma using the antibody-trapping method, in the presence of excess ox renin substrate. The renin-like activity after trypsin was indeed due to renin, since Ang I generation in dog plasma before and after trypsin treatment was completely inhibited by H-77 at 10(-6) mol/l, and the two IC50 values were very similar (2.7 +/- 0.7 and 2.9 +/- 0.7 at 10(-8) mol/l, respectively). Dog plasma inactive renin was effectively separated from active renin by chromatography on Affigel Blue. Like human prorenin, dog plasma inactive renin rose in response to sodium depletion (furosemide 5 mg/kg, i.v.) followed by a low-salt diet (1 mmol Na+/day) for 4 days, (from 29.6 +/- 8 to 162 +/- 22 microU/ml; P less than 0.01, n = 10). Active renin also increased as expected. Intravenous captopril (6 mg/kg per h), for 3 h, led to a sharp increase in dog plasma active renin (from 53 +/- 8 to 360 +/- 60 microU/ml; P less than 0.01, n = 6), whereas inactive renin remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Vascular and adrenal reninlike activity in chronically diabetic rats.

The aim of this work was to investigate, in an experimental model of diabetes mellitus, the levels of renin activity in vascular and adrenal tissues and their relationship to several circulating renin-angiotensin system components. Rats with chronic (12 weeks) streptozocin-induced diabetes showed a significant decrease in plasma renin activity (PRA), plasma renin concentration, and plasma aldosterone. However, plasma trypsin activatable inactive renin concentration was increased (11.65 +/- 1.40 vs 6.73 +/- 0.57 ng angiotensin I/ml/hr; p less than 0.001), as were aortic reninlike activity (p less than 0.001) and adrenal renin, both in the zona glomerulosa (p less than 0.01) and the fascicular-reticular-medullary portion (p less than 0.001) with respect to an age-matched control group. After bilateral nephrectomy, plasma renin-angiotensin system components (PRA and plasma active and inactive renin concentrations) as well as aortic and fascicular-reticular-medullary renin activity significantly decreased in both control and diabetic rats. However, glomerular renin activity increased in control nephrectomized rats to the levels observed in diabetic animals but did not change in diabetic nephrectomized rats. The parallel changes of aortic and fascicular-reticular-medullary renin activity and plasma inactive renin concentration in diabetes and nephrectomy suggest an interdependent relationship, whereas the increase of glomerular renin activity in diabetic and nephrectomized animals, both with low levels of PRA, suggests the existence of a local autonomic renin-angiotensin system regulated by plasma feedback. Tissue renin-angiotensin system alterations in diabetes could mean that a pathogenic factor is involved in long-term diabetic complications or that only a compensatory physiological process is at work.

Evidence for a predominant renal secretion and clearance of inactive plasma renin, studied by in vivo inhibition of protein synthesis

The aim was to investigate the contribution of the kidneys and the extrarenal tissues to the concentration of inactive renin in the circulation. Renin was determined in plasma from normal, conscious, male mice in strains with low (BALB/c) and high (Theiller) renin content in the submandibular glands. In untreated, conscious males, inactive and active renin concentration in plasma was 24.9 GU/l (range 13.6-40.1 GU/l) and 4.2 GU/l (0.6-8.8 GU/l), respectively in the BALB/c strain (n = 28). These values were not significantly different from the concentrations of inactive (29.2 GU/l, range 15.6-88.3 GU/l) and active (5.1 GU/l, range 1.9-10.4 GU/l) renin in Theiller's strain (n = 10). Thus, inactive renin comprised 80-90% of the total renin in plasma from normal, conscious, male mice of both strains. The percentage of inactive renin was similar to that found in humans. Following sialo-adenectomy and bilateral nephrectomy, the plasma concentration of inactive renin was unchanged. The effect of in vivo inhibition of the protein synthesis by intraperitoneal injection of emetine was also investigated. After 6 h of inhibition, inactive renin in plasma declined from 21.0 to 9.9 GU/l in normal mice. In contrast, inactive renin was unchanged in sialo-adenectomized and nephrectomized mice (29.1 versus 28.6 GU/l). Our findings suggest that the kidneys are the main source of inactive renin in the circulation. The data also indicate that the kidneys simultaneously secrete and remove inactive renin from the blood at approximately the same rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Reno-submandibular axis controls release of extrarenal inactive renin

The mechanisms causing the release of plasma inactive renin (PIR) area still unclear. We have investigated the role of the kidney in the release of trypsin-activable PIR from extrarenal sources in the rat, with special reference to the submandibular gland. The activation of PIR was performed by incubation with 20 mg/ml trypsin at 4 degrees C for up to 10 min; the reaction was then terminated by addition of 20 mg/ml of soybean trypsin inhibitor. Bilateral nephrectomy resulted in a gradual, marked, sex-independent increase in PIR concentration, reaching levels 4.5 times higher than basal in 24 h (time 0: 14.8 +/- 1.0 ng/ml per h; 24 h: 66.8 +/- 3.4 ng/ml per h, mean +/- s.d., P less than 0.001). This increase was not altered by the concomitant intravenous infusion of pressor doses of either angiotensin (Ang) II (30 ng/min) or pure mouse submandibular renin (a 20-ng intravenous bolus followed by intravenous infusion at the rate of 50 ng/h) for 4 h, but was completely prevented by prior removal of the submandibular glands, in which no activity of active renin and no inactive renin was detected. These results suggest that the post-nephrectomy increase of PIR is not dependent on feedback mechanisms of the suppressed renin-angiotensin system, but is controlled by the presence of submandibular glands in the rat.

Evidence for a predominant renal secretion and clearance of inactive plasma renin, studied by in vivo inhibition of protein synthesis, and inhibition of renal tubular protein reabsorption.

Inactive plasma renin is mainly secreted and eliminated by the kidneys in the mouse. The half-life of inactive plasma renin is 6 hours or less. The kidneys remove inactive renin from the circulation by glomerular ultrafiltration.

Effects of trypsin on the measurement of inactive renin in rat plasma by radio-immunoassay

We have examined the effect of trypsin treatment of rat plasma on the rate of angiotensin (Ang) I generation and measurement of this peptide by radio-immunoassay. Trypsin increased the renin incubation blank but did not alter the kinetics of the renin reaction with exogenous renin. The quantity of immunoreactive material detected in trypsin-treated plasma was not proportional to the volume of plasma assayed. Consequently, the level of inactive renin was dependent upon the volume of plasma subjected to the assay. This discrepancy occurred with two independent radio-immunoassay systems. The rate of Ang I generation was linear and significantly elevated following the addition of renin substrate to trypsin-treated plasma. However, if trypsin degradation of endogenous renin substrate was extensive and additional renin substrate was not provided, non-linear rates of Ang I generation occurred. Multiple additions of trypsin were necessary to activate maximally inactive rat plasma renin. Inactive renin accounted for 79 +/- 2% of the total enzyme activity in normal rats. Although active renin declined following bilateral nephrectomy, the ratio of active to inactive renin did not change. The data suggest that the kidney is the primary source of inactive renin in the normal rat.

Effect of cyclosporin on blood pressure and renin-aldosterone axis in rats.

Although hypertension is a frequent complication of cyclosporin A (CSA) therapy in clinical practice, little experimental information is available on the nature and the mechanism of this form of hypertension. We studied the effect of currently recommended therapeutic dosages of CSA, i.e., 5 (CSA5) and 20 (CSA20) mg.kg-1.day-1, on blood pressure and the renin-aldosterone system (RAS) in spontaneously hypertensive rats (SHR). Influence of in vivo CSA treatment on in vitro angiotensin II (ANG II)-stimulated aldosterone secretion by isolated adrenal glomerulosa cells (AGC) was also measured. CSA treatment in SHR resulted in a consistent increase in systolic blood pressure. This increase in blood pressure occurred in the absence of significant changes in creatinine clearance in CSA5 rats, whereas in CSA20 rats a significant reduction in creatinine clearance was observed. Sodium balance and serum calcium and magnesium concentrations were not different between the control group and either of the two CSA-treated groups of rats. Plasma renin concentration (PRC) and inactive renin (IR) were markedly elevated, but plasma renin substrate remained unchanged with CSA administration. Despite the presence of hyperreninemia, plasma aldosterone was not elevated, suggesting that CSA may induce relative adrenal resistance to ANG II. This possibility was tested using AGC isolated from CSA-treated rats. ANG II-stimulated aldosterone secretion in AGC was diminished by low dose and aborted by high dose CSA-treatment. Thus CSA administration in SHR induces a predictable increase in blood pressure in association with "hyperreninemic hypoaldosteronism."

Effects of angiotensin I converting enzyme inhibition and calcium channel blockade on plasma levels of active and inactive renin in conscious rabbits

The interplay of juxtaglomerular (jg) calcium fluxes and exposure to AII in the regulation of jg renin secretion, was examined in vivo. An inhibitor of angiotensin I converting enzyme (captopril), a blocker of calcium channels (verapamil) and AII amide were infused, singly or in combination, into the ear vein of conscious rabbits. The effects on arterial pressure, and on levels of active and inactive plasma renin were monitored. Captopril (50 μg · min −1 · kg −1) produced a greater percentage increase in renin secretion than did verapamil (20 μg · min −1· kg −1), whilst the percentage fall in arterial pressure was similar. AII amide counteracted more effectively the actions of captopril than those of verapamil. When captopril was infused first, addition of verapamil did not enhance renin secretion (P > 0.2). When verapamil was infused first, addition of captopril greatly enhanced renin secretion (P < 0.01). However, when captopril was infused first, and its actions then suppressed by AII amide, addition of verapamil led to extremely high rates of renin secretion. The findings suggest the following: (1) the short loop negative feedback plays in vivo an important role in the rapid modulation of jg renin secretion and the action of AII may involve up- and down-regulation at the receptor and/or post-receptor level; (2) infused agents have rapid access to the critical sites of jg cells; (3) exposure to raised concentrations to AII not only reduces the effectiveness of AII, but also enhances jg secretory responses to lack of AII, as well as to calcium channel blockade. Thus, at least some of the jg calcium channels appear to respond both to AII and to blockers; (4) extreme changes in the levels of active renin are possible without changes of inactive renin levels. Secretion of the latter may be under separate control, or its secretion rate parallelled by the rate of its activation.

Elevated levels of plasma prorenin (inactive renin) in diabetic and nondiabetic patients with autonomic dysfunction.

We measured plasma inactive renin (prorenin) levels in 46 diabetic patients, 4 nondiabetic patients with idiopathic autonomic dysfunction, and 115 normal subjects. Plasma inactive renin levels were normal in the diabetic patients who had no complications (n = 6) and in those with microvascular disease (n = 8) who did not have coexistent autonomic dysfunction. Plasma inactive renin was either grossly elevated or in the upper limit of the normal range in diabetic patients with autonomic dysfunction (n = 18). No correlation was found between plasma inactive renin and glycemic control, as measured by hemoglobin A1c. High plasma inactive renin levels were also found in the 4 nondiabetic patients with idiopathic autonomic dysfunction. These data suggest that increased plasma inactive renin levels in diabetic patients are a consequence of coexistent autonomic dysfunction. This finding is consistent with other evidence that suggests autonomic regulation of the processing of prorenin to renin within the kidney.

The cat: an animal model for studies of inactive renin.

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.

Activation of the renin system in acute pancreatitis

Activity of the renin-angiotensin system was assessed in patients with acute pancreatitis. Measurements of active plasma renin and inactive plasma renin were made in normal subjects, patients with acute pancreatitis, and patients with acute abdominal pain syndromes exclusive of pancreatitis. Active plasma renin values were significantly increased in acute pancreatitis, nearly 500 percent higher than in the other two groups. Inactive plasma renin values were similar in the three groups. In a subgroup of patients with acute pancreatitis, measurements were made on presentation and after recovery. The elevated active plasma renin values on admission fell significantly with recovery, in parallel with changes in serum amylase values. Inactive plasma renin values changed variably; there was a significant inverse regression relationship between the changes in active and inactive plasma renin values with recovery. The results indicate that the renin-angiotensin system is activated in acute pancreatitis to a significantly greater extent than in other syndromes with acute abdominal pain. The increased active plasma renin in acute pancreatitis is most likely due to renal release secondary to the reduced circulating volume and hypotensive effect of this disease. However, changes in the relationship between active and inactive plasma renin in some patients suggest that activation of inactive renin by proteolytic enzymes released in acute pancreatitis might play an additional role.

Quantitative activation and determination of inactive renin by high performance liquid chromatography.

Assay of inactive renin in unfractionated mouse plasma is difficult and often impossible because of high concentrations of active renin and plasma protease inhibitors. Therefore, 0.025 to 0.05 ml of plasma or amniotic fluid from mice was separated by high performance liquid chromatography (HPLC) using a silica-based size exclusion column. The eluates were examined for enzymatically active renin before and after limited proteolysis with trypsin. Since inactive renin eluted as a single peak corresponding to a molecular weight of 38,000 daltons and the elution of active renin was markedly retarded, inactive and active renin were partially separated. compared with plasma, inactive renin in amniotic fluid eluted as a broader and sometimes diphasic peak, suggesting heterogenicity. The rapid and reliable separation by HPLC provided a more than 300-fold purification of inactive renin. Despite low concentrations of plasma protease inhibitors, a 1 000 000-fold molar excess of trypsin (1 mg/ml) was needed for optimal activation. The necessity for high trypsin concentrations for activation may partly be explained by enzyme kinetic considerations. By combining HPLC with trypsin activation, inactive renin was readily measured and found to be 9.4 (5.1-13.2) GU (Goldblatt units)/l in normal and 8.0 (5.1-12.2) GU/l in sialoadenectomized and nephrectomized mouse plasma, which is higher than previously determined in our laboratory. The concentration was 12.4 (8.8-16.1) GU/l in amniotic fluid. Thus, the concentration of inactive renin in plasma is almost as high as active renin in normal mice (17.6 GU/l) and is uninfluenced by the decrease of active renin to 0.3 GU/l after sialoadenectomy and nephrectomy.

Activation by puff adder venom of inactive renin in normal and hypertensive rat plasma.

Inactive renin has been studied extensively in human plasma, but in animal plasma its accurate quantification has proved more difficult, due partly to higher activity of plasma protease inhibitors. Such activity in human plasma can be conveniently destroyed by a metalloprotease in Bitis arietans venom, with concommitant release of endogenous enzyme activities, such as plasma kallikrein, that then activate inactive renin. It was therefore of interest to look for inactive renin in rat and rabbit plasma using this approach, so providing, in addition, a comparison for the disparate data of other groups who have used trypsin or acid for activation. In both rat and rabbit plasma the proportion of inactive renin was 62% of total renin, whereas human plasma contained more inactive renin and a higher proportion, 82%. A higher concentration of venom was required for rat (33 ug venom/ml plasma) and rabbit (4 micrograms/ml) than needed for activation, at a similar rate, in human plasma (1 microgram/ml). When applied to studies of rats made hypertensive and hyper-reninaemic by aortic ligation for 5 days, higher total (active + inactive) renin was observed. The proportion of inactive renin, as a percentage of total renin in plasma collected at this time, was, however, found to diminish significantly. In conclusion, puff adder venom activates inactive renin in rat and rabbit plasma and can be used to study physiological changes in inactive renin in such animal plasma.

Inactive renin in peritoneal fluid of the cat: responses to acute stimuli which alter plasma renin.

Active and inactive renin in plasma and peritoneal dialysate were studied in pentobarbitone anesthetized cats during 8 hours of peritoneal dialysis. In control cats, despite a significant rise in plasma active renin from 1.3 +/- 0.22 to 4.4 +/- 0.76 pmoles AI/ml/hr over 8 hours, plasma inactive renin was unchanged. Four-fold rises in plasma active renin after hemorrhage (15 ml/kg) and captopril (1 mg/Kg I.V.) were unaccompanied by significant change in inactive renin. Bilateral nephrectomy resulted in undetectable plasma active renin after 3 hours but plasma inactive renin was 25-30% of initial levels 6 hours post-nephrectomy. In peritoneal dialysate, active renin remained low in control cats but there was a ten-fold rise of inactive renin from 0.06 +/- 0.02 to 0.60 +/- 0.07 pmoles AI/ml/hr. After hemorrhage there was a slight rise of active renin after 3 and 4 hours. Inactive renin was lower than control 6 hours after nephrectomy and captopril, but higher than control 1 and 2 hours after hemorrhage. Dialysate inactive renin accumulation slopes were not different among treatments. Acute changes in plasma active renin have little effect on peritoneal dialysate renin.

Roles of kidney and submandibular gland in the release of rat plasma inactive renin

In this study we outlined the development of an enzymatic technique to activate plasma inactive renin by trypsin in rat plasma. Using this method, we reported the releasing mechanism of the trypsin-activable inactive renin which has not yet been clarified. Adult male Wistar rats (260-300 g) were kept on regular diet (Na: 260 mg/100g) unless explained and underwent operation under pentobarbital anesthesia (50 mg/kg). Blood samples were obtained from conscious rats through the cannulae, which had been inserted into the left femoral arteries 24h before the experiments. After addition of excessive renin substrate which had been obtained from the 24 h-nephrectomized rat plasma, renin was measured by the commercial RIA-kit (Dainabot). Trypsin (Worthington) treatment (20 mg/ml plasma for 10 min at 4 degrees C) was followed by addition of SBTI (Sigma) (20 mg/ml plasma). This condition maximally increased the rate of angiotensin I generation and did not alter the Km or optimum pH of the renin reaction. In this condition, trypsin reaction was completely inhibited by adding these concentrations of SBTI. The molecular weight of inactive renin (51,000) in the normal rat plasma estimated by Sephadex G-100 column (Pharmacia) was the same as that in the nephrectomized rat plasma. In conclusion, trypsin treatment of plasma (20 mg/ml plasma for 10 min at 4 degrees C) followed by SBTI (20 mg/ml plasma) was justified for trypsin activation of rat plasma. Using this method, we investigated the changes in active and inactive renin after bilateral nephrectomy in the salt-depleted rat. Active renin decreased rapidly after bilateral nephrectomy with a half life of 23.6 +/- 4.0 min. Inactive renin, on the other hand, increased gradually and reached to a plateau 24 h after bilateral nephrectomy, and was kept unchanged during the following 24 h. The infusion of mouse submandibular gland active renin or angiotensin II could not prevent the increase of plasma inactive renin in the nephrectomized rat. These suggest that there may be no feedback mechanisms between plasma inactive and active renin or angiotensin II. Furthermore, we investigated the organ-related sources of plasma inactive renin which markedly increased after total nephrectomy. Simultaneous removals of submandibular glands but not of adrenal glands completely prevented the postnephrectomy increases of plasma inactive renin. But, removals of submandibular glands or adrenal glands alone were followed by no changes in the basal levels of plasma inactive renin.(ABSTRACT TRUNCATED AT 400 WORDS)

Active and Inactive Renin in the Cat

Pentobarbitone-anesthetized cats underwent peritoneal dialysis and had blood samples removed and kidneys deep frozen at sacrifice. Inactive renin is easily measurable in cat plasma and peritoneal dialysate fluid. Only small amounts are found after acid activation at pH 4.0, but large amounts after trypsin 2 mg/ml at 4 degrees C for 10 minutes. Mean active renin in pentobarbitone-anesthetized cats was 1.8 +/- 0.4 pmoles AI/ml/hr, while inactive renin was 2.3 +/- 0.5 pmoles AI/ml/hr. The increased angiotensin I producing activity after trypsin in peritoneal dialysate was most active at pH 7.0 (plasma and kidney active renin 7.25 and 7.85), and had an apparent molecular weight of 39-40,000. (Plasma active renin had an apparent MW of 33,500 and kidney active renin 36,000. Plasma inactive renin had an apparent MW of 35,500) Cat plasma after cibacron-blue affinity chromatography showed mainly active renin in the breakthrough buffer (30% of total renin eluted), and renin which is almost entirely inactive in the bound peak (70% of total renin eluted). Active renin from plasma and kidney, and activated inactive renin from concentrated peritoneal fluid, showed exactly similar inhibition by the renin inhibitor H77 (IC50 0.3 microM). Cat plasma angiotensinogen had an apparent MW of 53,000.

Potential Functions of Plasma Prorenin; Regional Activation and Tissue Extraction

The fate of circulating inactive prorenin was examined in patients and volunteers. Prorenin was activated either by acid-dialysis with warming at pH 3.3 or with trypsin. The results were similar but omission of warming reduced the value by 13%. In 6 volunteers, 20 min forearm venous occlusion raised regional total (T) and inactive (I) plasma renin concentration (PRC) by 51% and 48% without change of active (A) renin. During intense forearm exercise the ratio APRC: IPRC did not change in muscle or skin venous blood. Body anaerobic exercise increased APRC 3.7-fold without change in IPRC. These procedures activate plasminogen but are without effect on prorenin. In 18 patient with stable angina, TPRC was lower in coronary sinus than arterial blood (p less than 0.001) but APRC was not affected. A-V differences were not detected across the leg. Prorenin is apparently stable in the circulation but extracted by the heart.

Uteroplacental unit as a source of elevated circulating prorenin levels in normal pregnancy

Circulating levels of inactive renin, that is, prorenin, are increased in normal pregnant women. To determine whether the uteroplacental unit secretes prorenin into the maternal circulation, we measured enzymatically active and inactive renin in plasma simultaneously obtained from the radial artery and uterine vein of 12 normotensive, nonlaboring patients undergoing elective cesarean section at term. We also measured these forms of renin in the umbilical arterial and venous blood of these patients. Our data reveal that (1) the levels of inactive renin in both arterial and uterine venous blood of normal pregnant women are significantly higher than in peripheral venous blood of nonpregnant, normotensive control subjects; (2) normotensive term patients have a ratio of plasma inactive to active renin of 9:1 in contrast to the 1:1 ratio in normotensive nonpregnant subjects; (3) there is a significant uterine arteriovenous difference of prorenin (66.2 ± 24.4 ng/ml/hr, p < 0.05) but not of active renin (1.8 ± 1.5 ng/ml/hr, not significant). These results suggest that the uteroplacental unit contributes to the elevated prorenin levels at term pregnancy.

Analysis of inactive renin by renin profragment monoclonal antibodies

Two peptides were synthesized, corresponding to the sequences (−19 to −7) and (−26 to −17) of the prorenin prosegment. Monoclonal antibodies were raised to these sequences and used to characterize human plasma inactive renin. Only anti (−19 to −7) reacted with inactive renin, as measured by direct assay or affinity chromatography. The data were used to evaluate two possible inactive renin stuctures: (i) plasma inactive renin is a truncated prorenin lacking the prosegment N-terminal portion; (ii) its spatial conformation masks the N-terminal extremity, preventing interaction of this region with specific antibodies.

Human Plasma and Kidney Inactive Renin is Prorenin

Human Plasma and Kidney Inactive Renin is Prorenin