Receptor-mediated nonproteolytic activation of prorenin and induction of TGF-β1 and PAI-1 expression in renal mesangial cells

While elevated plasma prorenin levels are commonly found in diabetic patients and correlate with diabetic nephropathy, the pathological role of prorenin, if any, remains unclear. Prorenin binding to the (pro)renin receptor [(p)RR] unmasks prorenin catalytic activity. We asked whether elevated prorenin could be activated at the site of renal mesangial cells (MCs) through receptor binding without being proteolytically converted to renin. Recombinant inactive rat prorenin and a mutant prorenin that is noncleavable, i.e., cannot be activated proteolytically, are produced in 293 cells. After MCs were incubated with 10(-7) M native or mutant prorenin for 6 h, cultured supernatant acquired the ability to generate angiotensin I (ANG I) from angiotensinogen, indicating both prorenins were activated. Small interfering RNA (siRNA) against the (p)RR blocked their activation. Furthermore, either native or mutant rat prorenin at 10(-7) M alone similarly and significantly induced transforming growth factor-β(1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin mRNA expression, and these effects were blocked by (p)RR siRNA, but not by the ANG II receptor antagonist, saralasin. When angiotensinogen was also added to cultured MCs with inactive native or mutant prorenin, PAI-1 and fibronectin were further increased significantly compared with prorenin or mutant prorenin alone. This effect was blocked partially by treatment with (p)RR siRNA or saralasin. We conclude that prorenin binds the (p)RR on renal MCs and is activated nonproteolytically. This activation leads to increased expression of PAI-1 and transforming growth factor-β(1) via ANG II-independent and ANG II-dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may play a role in the development of diabetic nephropathy.     

Bovine ovarian cells have (pro)renin receptors and prorenin induces resumption of meiosis in vitro

The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10⁻¹⁰, 10⁻⁹, and 10⁻⁸ M incubated with oocytes co-cultured with follicular hemisections for 15 h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10⁻⁷, 10⁻⁵, and 10⁻³ M blocked this effect (P < 0.05). To determine the involvement of angiotensin II in prorenin-induced meiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200 μM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (P < 0.05). Only the oocytes’ cyclic adenosine monophosphate levels seemed to be regulated by prorenin and/or forskolin treatment after incubation for 6 h. To the best of our knowledge, this is the first study to identify the (pro)renin receptor in ovarian cells and to demonstrate the independent role of prorenin in the resumption of oocyte meiosis in cattle.     

High Salt Intake Damages the Heart through Activation of Cardiac (Pro) Renin Receptors Even at an Early Stage of Hypertension

Objective

It has not yet been fully elucidated whether cardiac tissue levels of prorenin, renin and (P)RR are activated in hypertension with a high salt intake. We hypothesized that a high salt intake activates the cardiac tissue renin angiotensin system and prorenin-(pro)renin receptor system, and damages the heart at an early stage of hypertension.

Methods

Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) received regular (normal-salt diet, 0.9%) and high-salt (8.9%) chow for 6 weeks from 6 to 12 weeks of age. The systolic blood pressure, plasma renin activity (PRA) and plasma angiotensin II concentration were measured, and the protein expressions of prorenin, (pro)renin receptor, angiotensinogen, angiotensin II AT1 receptor, ERK1/2, TGF-β, p38MAPK and HSP27 in the myocardium were investigated. The cardiac function was assessed by echocardiography, and histological analysis of the myocardium was performed.

Results

The high-salt diet significantly increased the systolic blood pressure, and significantly reduced the PRA and plasma angiotensin II concentration both in the WKYs and SHRs. Cardiac expressions of prorenin, renin, (P)RR, angiotensinogen, angiotensin II AT1 receptor, phosphorylated (p)-ERK1/2, p-p38MAPK, TGF-β and p-HSP27 were significantly increased by the high salt diet both in the WKYs and SHRs. The high-salt diet significantly increased the interventricular septum thickness and cardiomyocyte size, and accelerated cardiac interstitial and perivascular fibrosis both in the WKYs and SHRs. On the other hand, dilatation of left ventricular end-diastolic dimension and impairment of left ventricular fractional shortening was shown only in salt loaded SHRs.

Conclusion

The high-salt diet markedly accelerated cardiac damage through the stimulation of cardiac (P)RR and angiotensin II AT1 receptor by increasing tissue prorenin, renin and angiotensinogen and the activation of ERK1/2, TGF-β, p38MAPK and HSP27 under higher blood pressure.     

Combining angiotensin II blockade and renin receptor inhibition results in enhanced antifibrotic effect in experimental nephritis

The limited antifibrotic effect of therapeutic angiotensin blockade, the fact that angiotensin blockade dramatically elevates renin levels, and recent evidence that renin has an angiotensin-independent, receptor-mediated profibrotic action led us to hypothesize that combining renin receptor inhibition and ANG II blockade would increase the antifibrotic effect of angiotensin blockade alone. Using cultured nephritic glomeruli from rats with anti-Thy-1-induced glomerulonephritis, the maximally effective dose of enalaprilate was determined to be 10(-4) M, which reduced mRNAs for transforming growth factor (TGF)-β1, fibronectin (FN), and plasminogen activator inhibitor-1 (PAI-1) by 49, 65, and 56% and production of TGF-β1 and FN proteins by 60 and 49%, respectively. Disease alone caused 6.8-fold increases in ANG II levels that were reduced 64% with enalaprilate. In contrast, two- and threefold disease-induced increases in renin mRNA and activity were further increased 2- and 3.7-fold with 10(-4) M enalaprilate treatment. Depressing the renin receptor by 80% with small interfering (si) RNA alone reduced fibrotic markers in a manner remarkably similar to enalaprilate alone but had no effect on glomerular renin expression. Enalaprilate and siRNA combination therapy further reduced disease markers. Notably, elevated TGF-β1 and FN production was reduced by 73 and 81%, respectively. These results support the notion of a receptor-mediated profibrotic action of renin, suggest that the limited effectiveness of ANG II blockade may be due, at least in part, to the elevated renin they induce, and support our hypothesis that adding renin receptor inhibitor to ANG II blockade in patients may have therapeutic potential.     

(Pro)renin promotes fibrosis gene expression in HEK cells through a Nox4-dependent mechanism

The (pro)renin receptor (PRR) has recently been demonstrated to bind equally well renin and its precursor, prorenin, leading to a similar intracellular signaling independent of angiotensin II. In this study, we report that human embryonic kidney cells (HEK) exposed to renin or prorenin for 24 h in the presence of a blocking concentration of the angtiotensin-converting enzyme inhibitor perindoprilate increased superoxide anion production as measured by luminescence (lucigenin) and electron spin resonance spectroscopy (hydroxylamine radical transition). Also, both renin and prorenin increased Nox4 expression while Nox2, p47(phox), and p67(phox) remained unchanged. In an investigation of the effects of renin and prorenin on fibrosis genes, it appeared that both proteins stimulated transforming growth factor-β (TGF-β), fibronectin, and plasminogen activator inhibitor type 1 (PAI-1) expression and therefore participated to an overall switch toward a profibrotic state of the kidney cells. When the cells were transfected with a siRNA targeting the PRR, Nox4 expression was efficiently prevented as well as the increase in superoxide production, TGF-β, fibronectin, and PAI-1. Finally, we demonstrated that transfection of the cells with a Nox4-specific small interfering (si) RNA also prevented fibrosis gene expression following treatment with renin or prorenin. The results demonstrate that renin and prorenin, through their specific membrane receptor and independently of angiotensin II, promote fibrosis gene expression via a Nox4-dependent mechanism.     

Enhanced intrarenal receptor-mediated prorenin activation in chronic progressive anti-thymocyte serum nephritis rats on high salt intake

Despite suppression of the circulating renin-angiotensin system (RAS), high salt intake (HSI) aggravates kidney injury in chronic kidney disease. To elucidate the effect of HSI on intrarenal RAS, we investigated the levels of intrarenal prorenin, renin, (pro)renin receptor (PRR), receptor-mediated prorenin activation, and ANG II in chronic anti-thymocyte serum (ATS) nephritic rats on HSI. Kidney fibrosis grew more severe in the nephritic rats on HSI than normal salt intake. Despite suppression of plasma renin and ANG II, marked increases in tubular prorenin and renin proteins without concomitant rises in renin mRNA, non-proteolytically activated prorenin, and ANG II were noted in the nephritic rats on HSI. Redistribution of PRR from the cytoplasm to the apical membrane, along with elevated non-proteolytically activated prorenin and ANG II, was observed in the collecting ducts and connecting tubules in the nephritic rats on HSI. Olmesartan decreased cortical prorenin, non-proteolytically activated prorenin and ANG II, and apical membranous PRR in the collecting ducts and connecting tubules, and attenuated the renal lesions. Cell surface trafficking of PRR was enhanced by ANG II and was suppressed by olmesartan in Madin-Darby canine kidney cells. These data suggest the involvement of the ANG II-dependent increase in apical membrane PRR in the augmentation of intrarenal binding of prorenin and renin, followed by nonproteolytic activation of prorenin, enhancement of renin catalytic activity, ANG II generation, and progression of kidney fibrosis in the nephritic rat kidneys on HSI. The origin of the increased tubular prorenin and renin remains to be clarified. Further studies measuring the urinary prorenin and renin are needed.     

Measurement of inactive renin in normal, nephrectomized, and adrenalectomized rats.

In a new method for measurement of inactive rat plasma renin, the trypsin generated angiotensin I immunoreactive material, which was HPLC characterized as similar to tetradecapeptide renin substrate, is removed by a cation exchange resin before the renin incubation step. The method also corrects for trypsin destruction of endogenous angiotensinogen by the addition of exogenous angiotensinogen. When measured with this method inactive renin in rat plasma decreased after nephrectomy and increased after adrenalectomy. This is in accordance with findings in humans. A sexual dimorphism of prorenin (inactive renin) in rat plasma, similar to that reported in humans and mice, was demonstrated. Thus, inactive renin in the rat is no exception among species, and the rat might be a suitable animal model for further studies dealing with the physiology of prorenin in plasma and tissues.     

Increased renin excretion is associated with augmented urinary angiotensin II levels in chronic angiotensin II-infused hypertensive rats

Renin expression in principal cells of collecting ducts (CD) is upregulated in angiotensin II (ANG II)-dependent hypertensive rats; however, it remains unclear whether increased CD-derived renin undergoes tubular secretion. Accordingly, urinary levels of renin (uRen), angiotensinogen (uAGT), and ANG II (uANG II) were measured in chronic ANG II-infused Sprague-Dawley rats (80 ng/min for 14 days, n = 10) and sham-operated rats (n = 10). Systolic blood pressure increased in the ANG II rats by day 5 and continued to increase throughout the study (day 13; ANG II: 175 ± 10 vs. sham: 116 ± 2 mmHg; P < 0.05). ANG II infusion increased renal cortical and medullary ANG II levels (cortical ANG II: 606 ± 72 vs. 247 ± 43 fmol/g; P < 0.05; medullary ANG II: 2,066 ± 116 vs. 646 ± 36 fmol/g; P < 0.05). Although plasma renin activity (PRA) was suppressed in the ANG II-infused rats (0.3 ± 0.2 vs. 5.5 ± 1.8 ng ANG I·ml(-1)·h(-1); P < 0.05), renin content in renal medulla was increased (12,605 ± 1,343 vs. 7,956 ± 765 ng ANG I·h(-1)·mg(-1); P < 0.05). Excretion of uAGT and uANG II increased in the ANG II rats [uAGT: 1,107 ± 106 vs. 60 ± 26 ng/day; P < 0.0001; uANG II: 3,813 ± 431 vs. 2,080 ± 361 fmol/day; P < 0.05]. By day 13, despite suppression of PRA, urinary prorenin content increased in ANG II rats [15.7 ± 3 vs. 2.6 ± 1 × 10(-3) enzyme units excreted (EUE)/day, P < 0.01] as was the excretion rate of renin (8.6 ± 2 × 10(-6) EUE/day) compared with sham (2.8 ± 1 × 10(-6) EUE/day; P < 0.05). Urinary renin and prorenin protein levels examined by Western blot were augmented ～10-fold in the ANG II-infused rats. Concomitant AT(1) receptor blockade with candesartan prevented the increase. Thus, in ANG II-dependent hypertensive rats with marked PRA suppression, increased urinary levels of renin and prorenin reflect their augmented secretion by CD cells into the luminal fluid. The greater availability of renin and AGT in the urine reflects the capability for intratubular ANG II formation which stimulates sodium reabsorption in distal nephron segments.     

Possible activation of human prorenin by contact with the cell membrane

Semipurified human kidney prorenin was exposed in vitro to a mixture of lipids mimicking the composition of the inner leaflet of the cell membrane, in the presence of semipurified human angiotensinogen at a concentration of 1/4 Km. Prorenin was activated in a time-dependent manner over a period of 60 min. This lipid-dependent activation was completely reversed thereafter. Pre-incubation with anti-renin serum completely prevented this activated prorenin-dependent generation of angiotensin I. Our data suggest that human prorenin can be reversibly activated by contact with the cell membrane.     

Prorenin induces ERK activation in endothelial cells to enhance neovascularization independently of the renin-angiotensin system

Prorenin is an enzymatically inactive precursor of renin, and its biological function in endothelial cells (ECs) is unknown despite its relevance with the incidence of diabetic microvascular complications. Recently, (pro)renin receptor was identified, and the receptor-associated prorenin system has been discovered, whereas its expression as well as function in ECs remain unclear. In the present study, we found that ECs express the (pro)renin receptor, and that prorenin provoked ERK activation through (pro)renin receptor independently of the renin-angiotensin system (RAS). Prorenin stimulated the proliferation, migration and tube-formation of ECs, while it inhibited endothelial apoptosis induced by serum and growth factor depletion. MEK inhibitor abrogated these proangiogenic effects of prorenin, while AT1 receptor antagonist or angiotensin-converting enzyme inhibitor failed to block them. In vivo neovascularization in the Matrigel-plugs implanted into mouse flanks was significantly enhanced by prorenin, in which significant ERK activation was detected in ECs. Furthermore, tumor xenografts stably transfected with prorenin demonstrated the significantly accelerated growth rate concomitantly with enhanced intratumoral neovascularization. Our data demonstrated that the RAS-independent (pro)renin receptor-mediated signal transduction plays a pivotal role in the regulation of ECs function as well as in the neovascularization, and thus prorenin is potentially involved in the pathophysiology of diabetic microvascular complications as well as cancers.     

Human renin is correctly processed and targeted to the regulated secretory pathway in mouse pituitary AtT-20 cells

Renin is formed by intracellular processing of prorenin and catalyzes the conversion of angiotensinogen to angiotensin I, the precursor to angiotensin II. Several tissues synthesize prorenin. However, in man, the kidney is the only known source of circulating renin, raising the possibility that the processing enzyme is unique to that tissue. We have transfected a gene that directs prorenin synthesis in pituitary AtT-20 cells, which are capable of processing other prohormones. The results demonstrate that transfected AtT-20 cells can secrete inactive prorenin, accurately process prorenin to active renin, and be stimulated to release active renin in response to a secretagogue. These data imply that cellular elements capable of directing the processing of prorenin to renin and its correct subcellular compartmentalization may be present in nonrenal cell types and that critical elements of the regulated release of renin that occur in the kidney can be reconstituted in cells in culture.     

Prorenins activation by an enzyme from rat plasma (PreR-Co)

The aim of the present research was to explore the capacity of PreR-Co to process prorenin purified from kidney and corpora lutea (CL) and to study its action on extrarenal tissues. The PreR-Co was obtained from plasma as a single electrophoretic band by (NH4)2SO4 precipitation, gel filtration, anti-rat albumin immunoaffinity, and ion-exchange chromatography. Prorenin free of renin was obtained after (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography by a passage through an affinity gel of H-77 Sepharose. SDS-PAGE of supernatant and of acidic elution from gel, exhibited a single band of 43 kDa and 35 kDa, respectively; both recognized by the specific anti rat renin antibody. The isolated renin was not attacked by PreR-Co; on the contrary prorenin was completely activated. The product of PreR-Co-activated prorenin showed an analogous MW to that of renin and was recognized by the specific antibody. In addition to processing kidney prorenin, PreR-Co was able to cleave inactive renin from ovary, CL, uterus and adrenal gland homogenates. However, the amount of active renin generated from these tissues was lower than those produced by trypsin activation. PreR-Co is a good candidate for the role of the enzyme involved in tissues prorenin activation.     

High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin

Mannose-6-phosphate (man-6-P)/insulin-like growth factor-II (man-6-P/IgF-II) receptors are involved in the activation of recombinant human prorenin by cardiomyocytes. To investigate the kinetics of this process, the nature of activation, the existence of other prorenin receptors, and binding of native prorenin, neonatal rat cardiomyocytes were incubated with recombinant, renal, or amniotic fluid prorenin with or without man-6-P. Intact and activated prorenin were measured in cell lysates with prosegment- and renin-specific antibodies, respectively. The dissociation constant (K(d)) and maximum number of binding sites (B(max)) for prorenin binding to man-6-P/IGF-II receptors were 0.6 +/- 0.1 nM and 3,840 +/- 510 receptors/myocyte, respectively. The capacity for prorenin internalization was greater than 10 times B(max). Levels of internalized intact prorenin decreased rapidly (half-life = 5 +/- 3 min) indicating proteolytic prosegment removal. Prorenin subdivision into man-6-P-free and man-6-P-containing fractions revealed that only the latter was bound. Cells also bound and activated renal but not amniotic fluid prorenin. We concluded that cardiomyocytes display high-affinity binding of renal but not extrarenal prorenin exclusively via man-6-P/IGF-II receptors. Binding precedes internalization and proteolytic activation to renin thereby supporting the concept of cardiac angiotensin formation by renal prorenin.     

Chemical characterization of angiotensin I in porcine follicular fluid

In the search for novel neuropeptides in porcine follicular fluid (pff) using smooth muscle contractile activity as a response parameter, a substance with a marked activity was isolated in a pure form. By amino acid analysis and sequential study, this substance has been chemically revealed to be angiotensin I. A much smaller amount of additional activity was isolated and found to be angiotensin II, as determined by radioimmunoassay. Radioimmunoassays for angiotensin I and II confirmed that the amount of angiotensin I determined was much greater than that of angiotensin II. A comparative study of the extractions, however, indicated a large amount of angiotensin I had been generated from angiotensinogen by endogenous renin in the follicular fluid which could be activated during extraction and ultrafiltration at a low pH. These findings are consistent with the previous reports that described a high concentration of prorenin in the follicular fluid, acid activation of prorenin to renin and the subsequent generation of angiotensin I from endogenous angiotensinogen.     

Expression of (pro)renin receptor in human erythroid cell lines and its increased protein accumulation by interferon-γ

The renin-angiotensin system is known to enhance erythropoiesis. (Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, has recently been identified. However, expression of (P)RR in erythroid cells has not been studied. The aim of the present study is to clarify expression of (P)RR in erythroid cells, and the effects of erythropoietin, angiotensin II, transforming growth factor-β1 (TGF-β1), interferon-γ (IFN-γ) and interleukin-1β (IL-1β) on its expression. Western blot analysis showed that (P)RR protein was expressed in human cultured erythroid cell lines, YN-1 and YN-1-0-A (a clonal variant cell line of YN-1). Erythropoietin (1IU/ml) increased (P)RR mRNA expression levels in YN-1-0-A cells (1.7-fold increase compared with control), but angiotensin II did not. Treatment of YN-1-0-A cells with IFN-γ (10ng/ml) for 48h increased the expression levels of (P)RR protein significantly (1.4-fold increase compared with control), whereas it had no significant effects on expression levels of (P)RR mRNA. Treatment of YN-1-0-A cells with TGF-β1 or IL-1β for 24 or 48h had no significant effects on expression levels of (P)RR. The present study has shown for the first time expression of (P)RR in erythroid cells, raising the possibility that (P)RR may have a role in erythropoiesis and the pathophysiology of certain types of anemia.     

Activation and measurement of plasma prorenin in the rat.

Prorenin determination in rat plasma has been problematic from the outset. Consequently, its existence is questioned by some and its quantity by others, making it difficult for knowledge to advance as to its function relative to the renin system. The present study examines major variables in the determination of rat plasma prorenin and renin, notably different prorenin activation protocols involving blood samples obtained under various conditions from animals under different anesthetics. We found that a trypsin activation step with 5 mg/mL plasma, 60 min at 23 degrees C, followed by a PRA step of 10 min at 37 degrees C, resulted in the highest prorenin estimates, up to approximately 400 ng.mL-1.h-1 in terms of angiotensin I, as compared with published values of 0-190, based on other protocols. These estimates were obtained despite considerable destruction of angiotensinogen (renin substrate) by trypsin. Cryoactivation of prorenin was much less effective than in human plasma but, when followed by trypsin, it facilitated greater activation than with trypsin alone. Comparable fresh and fresh-frozen plasmas had similar prorenin-renin values, but lower values were observed in plasmas that had been repeatedly frozen and thawed. Conscious rats and those anesthetized with Inactin or ether had higher renins and prorenins than those anesthetized with methoxyflurane or halothane. Rats with kidneys in place during blood collection had higher renins (but not prorenins) than those whose kidneys were clamped off, suggesting that last-minute renin release during blood collection had occurred. We conclude that (i) trypsin generates increased renin, or renin-like, activity in plasma, suggesting activation of a precursor; (ii) on this basis, high prorenin levels exist in normal rat plasma; (iii) renin and prorenin levels are variously influenced by different anesthetics and blood handling procedures; (iv) variation in prorenin levels suggests that it is a dynamic (functional?) component of the renin system; (v) prorenin measurements are heavily influenced by methodological variations during the trypsin step or the subsequent PRA step; (vi) using standardized methodology, the rat can serve as a model for investigating the function of prorenin in normotension and hypertension.     

Measurement of renin and prorenin in cattle, hog and horse.

1. Species specific problems complicating the measurement of prorenin and renin concentrations were studied in bovine, hog and horse plasma. 2. In contrast to horse renin, bovine and hog renin reacted with rat angiotensinogen, allowing measurement of the plasma renin concentration in cattle and hog with rat angiotensinogen as exogenous substrate. 3. Trypsin treatment of plasma in order to activate prorenin generated an interfering angiotensin I immunoreactive material in all three species, most extensively in horse plasma. 4. This material could be removed in bovine and hog plasma by a cation-exchange resin, allowing an assay of the plasma prorenin concentration to be constructed in these species. 5. Another strategy has to be followed in order to measure prorenin and renin concentrations in horse plasma.     

Effect of oral contraceptives on the renin angiotensin system and renal function

We examined the effect of oral contraceptive (OC) usage on the renin angiotensin system (RAS) in two related experiments. In the first experiment, subjects were 34 healthy, normotensive, premenopausal women, 15 OC users and 19 OC nonusers, mean age 25 +/- 1 yr, ingesting a controlled sodium diet. We assessed arterial pressure, glomerular filtration rate, effective renal plasma flow, renal vascular resistance (RVR), and filtration fraction (FF) using inulin and p-aminohippurate clearance techniques, both at baseline and in response to the ANG II receptor blocker losartan. In the second experiment, in similar subjects, 10 OC users and 10 nonusers, we examined circulating RAS components [angiotensinogen, ANG II, aldosterone, plasma renin activity (PRA), and active renin] in response to incremental lower body negative pressure (LBNP), to determine whether renin secretion is suppressed by OC usage. OC users exhibited elevations in systolic blood pressure, RVR, and FF compared with nonusers, which were partially corrected by losartan. In the LBNP phase of the study, baseline measures of PRA, angiotensinogen, ANG II, and aldosterone were all increased in the OC group compared with the control group. Active renin levels did not differ between groups. Incremental LBNP resulted in increased circulating levels of RAS components in both groups. We conclude that the RAS is activated in women using OCs. There was no evidence that decreases in renin secretion result in normalization of the RAS as a whole.     

Identification of renal cathepsin B as a human prorenin-processing enzyme

Prorenin, the inactive biosynthetic precursor of renin, is proteolytically cleaved in the renal juxtaglomerular cells to renin. The activity of renin is rate-limiting for generation of angiotensin II in the circulation. We identified a renal thiol protease which activates and accurately cleaves the 43-amino acid prosegment of human recombinant prorenin. In the current studies, 6.5 mg of this protease was purified from human renal cortex using a three-step procedure dependent upon Leu-Leu-arginyl affinity chromatography. This represented an overall 766-fold purification and resulted in three protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of molecular weights 30,000, 25,000, and 24,000. All three bands cross-reacted with an anti-human liver cathepsin B antibody upon immunoblot analysis; electrolution of each band and amino-terminal sequence analysis confirmed that the Mr 30,000 protein was mature cathepsin B and the Mr 25,000 and 24,000 bands were cathepsin B subunits. The pH optimum for the hydrolysis of pure human recombinant prorenin by pure renal cathepsin B was 6, and the Michaelis-Menten constant, Km, of the reaction was 1.4 x 10(-9) M. Immunostaining of human kidney using a sheep anti-human cathepsin B antibody demonstrated the presence of cathepsin B in the juxtaglomerular areas of the kidney, as well as in the renal proximal tubules. Electron microscopic immunohistochemistry using the same antibody demonstrated cathepsin B in dense secretory granules of the juxtaglomerular cells. Renin was also shown to be present in these granules. This study provides both biochemical and morphological evidence that renal cathepsin B is a human prorenin-processing enzyme.