Form Of Renin Research Articles

Functional relevance of the microheterogeneity of active renin.

We previously demonstrated the presence of six active forms of renin in rats, based on their isoelectric points (pI). Intravenous or intraventricular injections yielded different renal effects with the different forms. Additional work is presented supporting the hypothesis that these forms are functionally different. Renin form 2 (pI = 5.7), form 4 (pI = 5.2) or form 6 (pI = 4.8) was infused intravenously (IV) in Spague-Dawley rats. The total renin infused over 30 minutes was the amount that could generate 1000 ng angiotensin I (Ang I)/h/100g body weight. Arterial blood pressure, urine volume, sodium, and potassium excretion were determined at 15-minute intervals. Plasma renin activity (PRA) and plasma aldosterone concentrations were measured 45 minutes after the start of infusion of renin forms. Infusion of renin form 4 increased urine volume and sodium excretion significantly. Renin forms 2 and 6 were without effect on renal excretion in spite of the fact that the same activity was infused. Both PRA and aldosterone concentrations after renin infusions were similar for all groups. Nonrenin renal protein with the same pI as form 4 was used as a control. This preparation was inactive and implies that our data with renin form 4 were not the result of contamination with a nonrenin protein having a pI of 5.2. In addition, the urinary responses could not be attributable to change in renal perfusion pressure as arterial blood pressure was not altered. These data support the hypothesis that there are functional differences among the different renin forms.

Prorenin and renin as separate mediators of tissue and circulating systems.

The renin gene is expressed in extrarenal tissues. High concentrations of renin occur in female reproductive organs: the adrenals of rats and mice have intermediate levels. The testis also synthesizes renin, as does the anterior pituitary. In contrast, very low levels of renin (ie, below plasma levels) are found in the heart and extrarenal vascular tissues. The predominant form of renin in the human ovary, placenta, and uterus is prorenin and the human ovary, testis, and adrenals have been shown to secrete prorenin into the circulation. The kidney is the only organ that secretes active renin and it is also the major source of plasma prorenin. We have used the ovary as a model to study extrarenal renin. Ovarian prorenin secretion changes dynamically during the menstrual cycle and during gestation. Secretion occurs during the LH surge and when hCG is present in the blood during pregnancy. Plasma active renin does not change concurrently and only low levels of active renin are found in association with very high concentrations of prorenin in ovarian follicular fluid after gonadotropin stimulation. To explain the preponderance of prorenin, and the virtual absence of renin in the ovary we hypothesize that prorenin need not be converted to active renin to have effect. In vitro, when prorenin is acidified to pH 3.3 or cooled to 0 degrees C, it develops intrinsic catalytic activity without cleavage of the prosegment.(ABSTRACT TRUNCATED AT 250 WORDS)

Processing of one-chain to two-chain renin in the mouse submandibular gland is influenced by androgen.

In the male CD-1 mouse submandibular gland (SMG) renin activity increases markedly with puberty. We have reported that this is, in part, due to an androgen-mediated increase in renin gene transcription. In this study, we examined whether posttranslational processing is also influenced by androgen. We studied secretion and processing of active renin in the CD-1 male mouse SMG which secretes primarily the Ren-2 renin isozyme before and after puberty and also compared findings with the adult female CD-1 mouse. Maturation increases renin level and secretion rate in the male SMG but much less in the female. In addition, Western blot analysis of molecular forms of renin in SMG tissue and media shows a predominance of 1-chain intermediate form of renin before puberty but of the 2-chain mature form thereafter. Adult female SMG contains and secretes much less active renin than the male organ. Administration of testosterone to the female CD-1 mouse induces an adult male level and pattern of secretion, with high concentrations of active 2-chain renin being secreted. These data suggest that SMG renin is androgen regulated, in part by an androgen-responsive enzyme that processes 1-chain renin to the 2-chain form.

Intrinsic Sympathomimetic Activity Counteracts Beta-Blocker Inhibition of Renin Activation

Enzymatically inactive renin (IR) is the predominant circulating form of renin. Sympathetic activity may influence plasma renin activity (PRA) by regulation of the conversion of IR to active renin (AR, PRA). It has been demonstrated previously that beta blockade lowers PRA at least partly through inhibition of this conversion process. The authors hypothesized that beta blockade and intrinsic sympathomimetic activity (ISA) would have opposing effects on production of AR from its inactive precursor. Eighteen primary hypertensives (12 male, 6 female, mean age 57.7 +/- 2.7) were entered in a placebo-controlled, double-blind crossover study of the effects of equipotent doses of pindolol and propranolol on mean +/- SEM systolic BP, diastolic BP, heart rate, active renin (AR), total renin (TR), inactive renin (IR), and % AR/TR. Drug dose was titrated to achieve a goal DBP of 90 mmHg or less. Active renin was defined as the rate of generation of angiotensin I in 37 degrees C plasma at pH 5.7. Total renin was determined by preincubation of plasma aliquots with 1.5 mg/mL trypsin in the presence of 5 mM benzamadine for one hour at -4 degrees C prior to assay of renin activity. Inactive renin was calculated as TR minus AR. The BP responses achieve by dose titration of propranolol and pindolol were virtually identical at rest, indicating equivalent depressor effects of the two beta blockers. Heart rate and active renin were, however, lowered to a much greater extent with propranolol as compared with pindolol. The lack of significant pindolol-induced fall in % AR/TR suggests that this drug has little net effect on the formation of AR from IR.(ABSTRACT TRUNCATED AT 250 WORDS)

Differently glycosylated multiple forms of renin in the blood circulation of rats.

Serial lectin chromatography of rat plasma on concanavalin A and lentil lectin columns separated plasma renin into differently glycosylated multiple forms. To study the role of glycosylation in the clearance of circulating renin, three different forms of glycosylated renin (renin A, B-1 and B-2) were highly purified from rat kidneys. Renin A and B-1 were loosely and tightly, respectively, bound to concanavalin A, but did not bind to lentil lectin. Renin B-2 was bound to both concanavalin A and lentil lectin. Each form of renal renin, labelled with 125I and intravenously given to rats, disappeared from the blood circulation at different rates, and distributed in the liver and kidney to different extents. Deglycosylation of renin B-1 with endoglycosidase F led to reduced metabolic clearance rates and to a significant decrease in uptake by the liver. These results show the heterogeneity in the fate of circulating renin due to the heterogeneity of glycosylation.

Cerebrospinal fluid angiotensin II in patients with essential hypertension.

We measured components of the cerebrospinal fluid renin-angiotensin system from patients with essential hypertension under different dietary sodium intakes. The cerebrospinal fluid concentration of angiotensin II (Ang II) from the patients on a normal-sodium diet was 1.36 +/- 0.41 fmol/ml (n = 5). Neither the inactive nor the active form of renin was detected by the enzymatic activity or by the immunoreactivity, whereas angiotensinogen was detected (38.6 +/- 3.1 pmol/ml, n = 5). The Ang II level remained unchanged even after incubation of the cerebrospinal fluid at 37 degrees C for 3 h. Further, when authentic Ang II was added to the cerebrospinal fluid followed by incubation for 3 h at 37 degrees C, more than 90% of the added Ang II remained unchanged. Thus, the cerebrospinal fluid Ang II level may be reflected by the activity of the brain Ang II-forming system, as it was not affected by the cerebrospinal fluid constituents. The circulating renin-angiotensin system was stimulated by sodium depletion, and the cerebrospinal fluid concentration of Ang II also increased significantly. Sodium depletion may stimulate the brain Ang-II forming system, as it does the circulating renin-angiotensin system.

Importance of glycosylation for hepatic clearance of renal renin.

Three differently glycosylated forms of renin (renin A, B-1, and B-2) were highly purified from rat kidneys by pepstatin-aminohexyl-Sepharose affinity chromatography and by serial lectin affinity chromatography on concanavalin A (con A) and lentil lectin-Sepharose, and the role of glycosylation of renin was investigated. Renin A and renin B-1 were loosely and tightly bound to con A, respectively, but did not bind to lentil lectin. Renin B-2 bound to both con A and lentil lectin. These three forms of renin were all similar in their physicochemical characteristics, including molecular weight, isoelectric point, specific activity, Km, optimum pH, and antigenicity. Each form of renin, labeled with 125I and given intravenously to anesthetized rats, disappeared from the circulation at different rates (metabolic clearance rates of 5.05 +/- 1.02, 17.1 +/- 2.5, and 36.0 +/- 4.1 ml.min-1.kg-1 for renins A, B-1, and B-2, respectively). Labeled renin A distributed to a similar extent in the liver and kidney (21.2 +/- 0.2 and 15.2 +/- 0.8% of the injected dose, respectively), whereas renins B-1 and B-2 were distributed predominantly in the liver (56.3 +/- 1.2 and 72.3 +/- 3.7% of the injected dose, respectively) and to a lesser extent in the kidney (4.3 +/- 0.3 and 2.1 +/- 0.2%, respectively). Deglycosylation of renin B-1 with endoglycosidase F resulted in no loss of its enzymatic activity or antigenicity but greatly reduced the metabolic clearance rate to 18% (from 17.1 +/- 2.5 to 3.09 +/- 0.17 ml.min-1.kg-1). Deglycosylation of renin B-1 greatly decreased its uptake by the liver (from 56.3 +/- 1.2 to 3.3 +/- 0.2%) and increased its uptake by the kidney (from 4.3 +/- 0.3 to 23.9 +/- 0.9%). These studies indicate the importance of glycosylation of renin for its hepatic uptake and metabolic clearance rate.

Renin in the female reproductive system.

The female reproductive system is characterized by the presence of local renin-angiotensin systems within the ovary and within the uterine lining. In the human ovary, renin arises from theca cells. In the uterus, the endometrium (uterine lining in the nonpregnant state) and the decidua (uterine lining in the pregnant state) are the major sources of renin. The primary form of renin produced by these extrarenal sources is prorenin. Angiotensin II, the active component of the renin cascade has a number of potential roles in the ovary and uterus. A key observation is that the human ovum and fetus is bathed in fluids rich in prorenin and angiotensin II.

Multiple renin forms in the adrenal gland.

Renin heterogeneity has been described in rat kidney and plasma. In this study, we used the isoelectric focusing method to 1) characterize the adrenal renin forms in control rats, in rats on low- and high-Na diets, and in nephrectomized rats; and 2) examine their resemblance with plasma renin. Active renin (AR) and inactive trypsin-activatable renin (IR) were measured in adrenal homogenates and plasma. Aliquots were subjected to isoelectric focusing gels. Activation with trypsin (5 mg/ml) was performed before or after isoelectric focusing. Results showed that adrenal glands contained AR and IR. The content of adrenal AR increased significantly only in rats fed a low-Na diet. Following anesthesia, nephrectomy, or high-Na intake, the content of adrenal AR and IR was not significantly changed. In plasma, an inverse relationship between AR and IR was found. Adrenal glands contained six forms of AR focusing at the same pH as those of plasma AR but in different proportions. After activation of IR in adrenal glands, two additional renin forms focusing at pH 6.4 and 6.1 were found, whereas after activation of plasma IR, two peaks focusing at pH 5.9 and 4.8 were significantly enhanced. Adrenal AR forms were modified by alterations of salt and water balance differently than plasma AR. These results support the hypotheses of an endogenous production of renin forms by the adrenal gland.

Renin in human fetal lung--a biochemical and immunohistochemical study.

Human fetal lung homogenates contain an inactive form of renin which may be revealed by trypsin treatment. When activated, this form of renin has some biochemical similarities with fetal kidney renin: the pH optimum of fetal lung renin is approximately 6.5; it is bound by Affigel Blue affinity chromatography resin; and is inhibited by a monoclonal antibody (R-3-36-16) raised to human kidney renin. Inactive renin, partially-purified from both fetal kidney and lung, differs from this in that the renal form is of low-molecular weight (LMW, 45,000 daltons), whereas that from fetal lung is of high molecular weight (HMW, 58,000 daltons). Using a sensitive alkaline phosphatase-anti-alkaline phosphatase (APAAP) procedure with a polyclonal anti-renin antibody (R-15), immunoreactive renin in fetal lung was found in vessels in mesenchyme between airways. The pattern of staining was distributed similarly to Factor VIII-related antigen, suggesting localization in endothelial cells.

Molecular biology of the renin-angiotensin system.

This paper reviews the molecular biology of the renin-angiotensin system. The renin gene structure is analyzed in detail, including an examination of the putative regulatory regions. The combined action of these regulatory sequences would result in the complex, tissue-specific expression and regulation observed in vivo. The expression of the tissue renin-angiotensin systems, which may have important physiological functions, is also described. In addition, the pathway of renin biosynthesis and secretion is reviewed. This includes speculation on the fate of circulating prorenin and the physiological role of multiple renin forms and secretory pathways. The molecular approaches described in this paper have greatly advanced our knowledge of the biology of the renin-angiotensin system. Future studies using these and other approaches should provide further insight into this complex system.

A pepstatin-immunometric assay for active renin in human plasma

Renin (EC 3.4.23) is a rate-limiting enzyme in the renin-angiotensin system that regulates the water-electrolyte balance and blood pressure. Plasma renin has been assayed by measurement of plasma renin activity (PRA), because purification of human renin is very difficult and no antibody has been available for direct radioimmunoassay of human plasma renin. Recently, some groups, including ours, purified human renin [l-3] and developed direct radioimmunoassays for human plasma renin [4-61. These assays were very sensitive but detect not only active but also inactive forms of renin. To develop a specific radioimmunoassay for active renin, a monoclonal antibody specific for active has been developed [7-lo]. Another way to develop a specific assay of active renin is to use renin inhibitor as a ligand. Pepstatin-A is a synthetic inhibitor specific to aspartyl proteases including renin [ll] and, as far as we know, renin is the only aspartyl protease in human plasma. In this preliminary study we developed a new immunometric assay that was specific for the active forms of renin in human plasma. For this purpose we used solid-phased pepstatin as a ligand to separate active renin from inactive renin.

Heterogeneity of glycosylation of circulating active and inactive renin.

Concanavalin A chromatography was used to examine rat plasma and the incubation medium of rat renal cortical slices, the objective being to assess the heterogeneity of glycosylation of active and inactive renin. Inactive renin was measured by activation with trypsin. Concanavalin A chromatography could separate both active and inactive renin in the plasma into three forms, including the unbound form, the loosely-bound form and the tightly-bound form, thereby suggesting the presence of differently glycosylated forms of active and inactive renin in the plasma. Rat renal cortical slices secreted all these three forms of active and inactive renin, hence these forms are mainly of renal origin. These results suggest that differently glycosylated forms of active and inactive renin are released from the kidney into the blood circulation.

Dosage immunoradiométrique de la rénine active et de la prorénine plasmatiques au cours des cycles stimulés pour FIVETE ou GIFT

Prorenin (PR) an inactive high molecular weight form of renin normally circulates in human plasma at a concentration of about 10 times that of active renin (AR) and this proenzyme seems to be linked to the reproductive function. It has been demonstrated that AR and PR are present at high concentrations in follicular fluid when the ovaries are stimulated with gonadotropins and that the PR plasma levels increase steadily after hCG injection with a correlation between blood PR and the number of developing follicles and corpora lutea. From September 1986 we studied the profile of immunoreactive active renin (AR) and prorenin (PR) in plasma during hyperstimulated cycles for IVFET or GIFT. All women were treated with a protocol combining GnRH analog (Decaptyl Ipsen Biotech, Paris France) and human menopausal gonadotropins until injection of 5,000 IU hCG. AR has been assayed in frozen samples by specific immunoradiometry (Renin RIA code 79 795, Pasteur Diagnostic, France) using two complementary monoclonal antibodies. A second assay of total renin was carried out after trypsin activation which revealed the inactive form. Progesterone (P), estradiol (E2) were measured by radioimmuno-assays. During the follicular phase, from day 1 of hMG administration to the day before hCG, no significant difference could be found between two groups, 63 pregnant or 60 nonpregnant cycles matched for age and number of oocytes retrieved, for E2, P, PR and AR. During the periovulatory period (D - 1, Do = day of hCG injection and D 1) no difference could be found for E2, P and PR (tabl. 1). In the 2 groups the mean E2 levels increased after hCG injection, as well as P and PR. But a significant difference appeared for AR which increased in the plasma immediately after hCG administration in the pregnant group whereas it decreased in the non-pregnant group (+2.5 vs -2 pg/ml) the mean variation between Do and D + 1 being significantly different in fertile cycles and in nonfertile cycles.(ABSTRACT TRUNCATED AT 400 WORDS)

Ovarian prorenin-renin-angiotensin system.

Renin is classically considered to be an enzyme that is synthesized by the kidneys and secreted into the circulation where it affects angiotensin production. We review here recent evidence that suggests the existence of an extra-renal renin system, the ovarian prorenin-renin-angiotensin system, which may be linked to reproductive function. Prorenin, the inactive form of the enzyme renin, is present in the fluid of mature human ovarian follicles in extremely high concentrations; however, only 1 per cent of the renin in follicular fluid is in the active form. Plasma prorenin increases about 2-fold at midmenstrual cycle at the time of the LH surge, and the peak of prorenin is sustained for about 2 days. No change in plasma active renin levels occurs at this time. Administration of hCG to women whose ovaries have been stimulated with gonadotropins results in much higher plasma prorenin levels and the height of prorenin response is directly related to the number of mature follicles. Plasma prorenin also increases 10-fold during the early stages of pregnancy. It begins to rise on days 8 to 12 after embryo transfer, in parallel with the rise in endogenous hCG. The ovaries are the apparent source of the increase in plasma prorenin in early pregnancy since no such increase in prorenin occurred in a woman with ovarian failure who conceived after receiving a donor egg. These results suggest that prorenin is synthesized and secreted by the mature ovarian follicle and by the corpus luteum in response to LH/hCG. They also suggest that an ovarian renin system exists that is regulated by changes in prorenin.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium, the renin-aldosterone system, and the hypotensive response to nifedipine.

Ionic, hormonal, and blood pressure responses to a single oral dose of the calcium channel blocker nifedipine were assessed in 25 essential hypertensive subjects. When grouped according to their renin-sodium profile, low renin subjects had a greater hypotensive response to nifedipine (change in diastolic blood pressure -20.0 +/- 1.4 vs -6.4 +/- 1.0%; p less than 0.005) than did high renin hypertensive subjects. The initial level of serum ionized calcium predicted the blood pressure response to nifedipine (r = 0.70, p less than 0.001), as did the initial plasma renin activity (r = 0.65, p less than 0.005). Nifedipine induced a transient rise in serum ionized calcium (from 2.22 +/- 0.02 to 2.28 +/- 0.02 mEq/L; p less than 0.01), while plasma renin activity was consistently elevated compared with initial values at 30 (p less than 0.01), 60 (p less than 0.01), and 120 (p less than 0.05) minutes after drug administration. By comparison, plasma aldosterone levels did not rise and even declined at 30 (p less than 0.01), 60 (p less than 0.05), and 120 (p less than 0.05) minutes after nifedipine. These results suggest that low renin hypertension is more critically dependent on extracellular calcium than are higher renin forms and demonstrate that levels of serum ionized calcium, plasma renin activity, or both may predict the sensitivity of blood pressure to calcium channel blockade. Lastly, calcium may play a pivotal role in vivo in coupling renin stimulation to adrenal aldosterone responses.

Multiple forms of immunoreactive renin in human adrenocortical tumour tissue from patients with primary aldosteronism.

There is increasing evidence which suggests that the adrenal gland contains the renin-angiotensin cycle. The localization of renin has been reported to be mainly in the zona glomerulosa rather than the fasciculata medullary portion. In the present study we have investigated extracts from aldosteronomas (n = 3), which are believed to derive from the zona glomerulosa cells. In addition, we have attempted to characterize the biochemical properties of the adrenal renin. Sizable quantities of renin-like activity (32.0 +/- 7.7 ng of angiotensin I generated h-1 mg-1 of protein, mean +/- SEM) were detected in the extracts. This renin-like activity was inhibited by anti-renin antibody raised against pure renin (mean, 95% of the total renin-like activity), indicating that it was not due to the non-specific action of proteases such as cathepsin D. The optimum pH of the tissue renin-like enzyme was 6.0 for rat plasma substrate. Differences were found, however, in the molecular mass (36,000, 37,000, 44,000 and 48,000), binding to concanavalin A and isoelectric points (4.40, 4.68 and 5.00). These results confirm the existence of specific renin in aldosteronoma. Renin microheterogeneity could be evidence for local production of the enzyme.

Two-step prorenin-renin conversion. Isolation of an intermediary form of activated prorenin.

Prorenin is an inactive form of the aspartic protease renin. Like pepsinogen, it is activated at low pH. The kinetics of acid activation of prorenin were studied in human amniotic fluid and plasma and in preparations of purified prorenin isolated from amniotic fluid and plasma. Conversion of prorenin (pR) into active renin (R) appeared to be a two-step process involving the generation of an intermediary form of activated prorenin (pRa). The pR----pRa step is an acid-induced reversible change in the conformation of the molecule, and the pRa----R step is proteolytic. pRa----R conversion occurred in amniotic fluid at low pH by the action of an endogenous aspartic protease. In plasma pRa----R conversion occurs after restoration of pH to neutral and is caused by the serine protease plasma kallikrein. pRa----R conversion did not occur in purified preparations of prorenin. Thus, in contrast to pepsinogen, the acid-induced reversible conformational change is not followed by autocatalysis. pRa of amniotic fluid and plasma could be separated from R by affinity chromatography on Cibacron blue F3GA-agarose, and R but not pRa was detected by an immunoassay using monoclonal antibodies reacting with R and not with pR. The first-order rate constant for pR----pRa conversion depends on the protonation of a polar group (or groups) with pK approximately 3.4, the rate constant being proportional to the fraction of pR molecules that have this group protonated. This is analogous to the reversible acid-induced conformational change of pepsinogen that occurs before its proteolytic conversion into pepsin. kcat/Km for pRa----R conversion by plasmin and plasma kallikrein at pH 7.4 and 37 degrees C was 7.8 X 10(6) and 5.2 X 10(6) M-1 min-1, respectively, which was about 50-70 times greater than for pR----R conversion. The susceptibility of pRa to proteolytic attack is high enough for the intrinsic factor XII-kallikrein pathway to cause rapid pRa----R conversion at 37 degrees C even in whole blood with its abundance of serine protease inhibitors. Formation of pRa may occur in vivo in an acidic cellular compartment, such as exo- or endocytotic vesicles.

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.

Secretion control for active and inactive renin: effect of ouabain on release from rabbit kidney cortex slices.

Release of active and inactive renin by rabbit kidney cortex slices was investigated. Inactive renin was estimated as the increase in renin activity after acidification (pH 2.8) of slice supernatant solutions. For kidney slices incubated in complete Krebs bicarbonate buffer, the Na-K-ATPase inhibitor ouabain (100 microM) reduced the secretion of both active (-19.2%) and inactive (-78.9%) forms of renin. In low Na buffers ([Na+] = 23 mM) active renin release was increased and inactive renin was suppressed. Both of these changes were abolished by addition of ouabain (100 microM). The reduction in inactive renin secretion produced by ouabain in complete Krebs buffer did not occur in low [Na+] buffers. In zero Ca2+ buffers containing EGTA (5 mM), secretion of both active and inactive renin was increased but these changes were abolished by addition of ouabain (100 microM). Incubating kidney slices in low Na+, zero Ca2+ media revealed differences between the secretion control mechanisms for the two forms of renin. The separate stimulatory effects of low Na+ and low Ca2+ were not additive for the release of active renin and inclusion of ouabain resulted in similar secretion rates to those under control conditions. For inactive renin secretion, in the absence of Ca2+ release mechanisms still respond to reduction in Na+ with decreased secretion. Conversely, in low Na+ buffers, removal of Ca2+ still promotes inactive renin secretion. These changes were abolished by the addition of ouabain (100 microM). Slices did not change in weight during incubation in media which did not contain ouabain. Addition of this inhibitor to control buffers and low Na buffers did result in an increase in weight. This correlated with the presence of Ca2+ in the buffer and did not appear to be related to [Na+]. These studies again show that the mechanisms regulating the secretion of active and inactive renin are not identical and support the hypothesis that Na+ have differing roles to play in the regulation of these two forms of renin.