Kinetic comparisons of amniotic fluid inactive renin and renal renin using synthetic and human renin substrates

Inactive renin has been isolated from pooled amniotic fluid and purified approximately 642-fold. Prior to activation the isolates had approximately 4% of the activity found after activation. The observation is similar to that reported for inactive renin from chorionic cell culture and suggests a placental origin of amniotic fluid inactive renin. Using plasma from an estrogen-treated woman, renin substrate was recovered free of renin and inactive renin and a portion was separated into NMW and HMW components. The NMW form constituted approximately 93% and the HMW form approximately 7% of the renin substrate. Amniotic fluid inactive renin was used for determinations of enzyme-substrate kinetics with the pooled, NMW, and HMW plasma substrate and tetradecapeptide synthetic substrate, and the results were compared to similar determinations using standard renal renin. Using synthetic substrate, the kinetics of renal renin and amniotic fluid inactive renin before and after activation were similar. The kinetics of renal renin with pooled, NMW, and HMW plasma substrate were also similar. Amniotic fluid inactive renin had a lower Km with pooled than with NMW substrate, however, which resulted from a significantly smaller Km with HMW component. Although the affinity constants with pooled substrate were not different for renin and inactive renin, the Km of inactive renin was significantly less with the HMW component of plasma renin substrate. The observations are compatible with a role for placental inactive renin in normal pregnancy and suggest the possibility of a further role in hypertensive pregnancy.     

High molecular weight renin identified in cytosol fractions of mouse renal cortices

In an attempt to confirm that high molecular weight renin was indeed true renin, we used a specific renin antibody and high performance liquid chromatography to determine characteristics of this protein. In mouse renin granules, renin was stored in a low molecular weight form of 38,000 daltons (LMW renin) and this molecular weight remained unchanged with application 20 mM of sodium tetrathionate. In the cytosol fraction of the renal cortex, LMW renin was partially converted to high molecular weight renin (HMW renin) of 65,000 daltons, as determined using tetrathionate. In both the LMW and HMW renin, enzymatic activity was completely neutralized by application of a specific antiserum of renin and an absolute amount of renin was identified by direct radioimmunoassay. The Km values of HMW and LMW renin were similar. Thus, LMW renin probably binds with renin binding substance and forms HMW renin.     

Renin: structural features of active enzyme and inactive precursor

To determine the structural basis for the unique catalytic mechanism of renin and the mechanism of activation of inactive renin, renin and inactive renin were isolated in pure form. The active site of renin consists of two aspartyl residues, two tyrosyl residues, and one arginyl residue, analogous to pepsin and other acid proteases. The complete amino acid sequence of mouse submaxillary gland renin was determined. Of the amino acids, 43% were identical to those in porcine pepsin. Combination of various chromatographic techniques permitted the separation of inactive renin from active renin in human plasma and kidney. Inactive renin of hog kidney was completely purified. Inactive renin consists of a single polypeptide chain and is activated by proteolysis but not by dissociative reagents such as 4 M NaCl or detergent. Thus it was concluded that the inactive renin in these tissues is renin zymogen rather than a renin-inhibitor complex.     

Correlation between juxtaglomerular index, kidney renin content and plasma renin concentration in the rat.

The correlation between juxtaglomerular index, kidney renin content, and plasma renin concentration has been investigated in rats. The results indicate that renin exists in two forms. When determining the renin content of the kidney, the renin actually present in the modified smooth muscle cells of the juxtaglomerular apparatus is measured; this is called bound renin. The amount of bound renin is derived from the total of granular and subgranular renin in the modified smooth muscle cells. Since JGI and KRCont show a significant positive correlation in untreated adult rats, it is assumed that in such animals the ratio of granular and subgranular renin is constant. Since no correlation could be demonstrated between kidney renin content and PRC in untreated adult rats, and JGI and KRCont did not change parallel with the increase of PRC in numerous experimental conditions, it is assumed that part of the renin synthetized in the JG cells is secreted directly, without passing the process of condensation into membrane bound granules. This mobile renin does not significantly affect the renin content and the JGI of the kidney. Under physiological circumstances, most of the produced renin seems to mature to granules in the modified smooth muscle cells before being secreted. When renin production and release increased, maturation to granules may be inhibited, a significant part of the produced renin released by direct secretion, and the subgranular, immature renin may also be secreted.     

Human renin activation by protease from the renin granule fraction of the dog kidney cortex

To clarify the possible conversion of prorenin in renin granules where conversion reportedly occurred, we investigated whether the renin granule fraction of the kidney could activate prorenin to the active form. Renin granules were isolated from the dog kidney cortex by discontinuous sucrose density gradient centrifugation. Human active renin was quantified by immunoradiometric assay which could detect only the human active renin but not the inactive human renin or dog renin. Inactive renin from human amniotic fluid was incubated with the subcellular fraction of the dog kidney cortex. The renin granule fraction that showed the highest renin activity stimulated the inactive renin to become the active form. The membrane preparation obtained from the renin granule fraction by freezing and thawing the fraction in low osmolarity retained the activity of renin activation. Other subcellular fractions showed less renin activation. The optimal pH for renin activation by the membrane was pH 5.0 to 6.0. The activation depended on the time of incubation and concentration. The activation was inhibited by N-ethylmaleimide but not by EDTA or serine protease inhibitors. These results suggest that renin is processed by a membrane bound protease in renin granules.     

Investigation of human and rat inactive renin in plasma and kidney.

Under an initial interval of immobilization stress in rats, reciprocal changes of plasma active and inactive renin were observed, suggesting activation of circulating inactive renin. Molecular weight (MW) studies revealed that this activation might proceed via a MW shift from inactive renin with MW of 50,000 to active renin of MW 43,000. In a later interval of stress, under stimulated renin secretion, a lower MW form (38,000) of active renin was released into the circulation. This MW is close to that of active renin (39,000) found in rat kidney renin granules. In renin granules, equilibrated in fractions of 1.6 and 1.7 mol/L sucrose in discontinuous density gradient, trypsin-activatable renin activity formed 36 and 16% of total activity, respectively. In humans, under acute bicycle exercise, a lower MW form (39,000) of active renin was released into the circulation, while the content of inactive renin with MW in the range of 51,000-58,000 and at 47,000 did not substantially change. There was a slight decrease in circulating inactive renin passing through the kidney. The data suggest that, at least in rats, in vivo pathways for activation of inactive renin might exist, other than that proceeding before secretion from renin granules. Under the conditions of increased renin secretion, a lower MW form of active renin is mainly released into the circulation in both rats and humans.     

Molecular cloning and nucleotide sequence of a human renin cDNA fragment.

We have studied human renin messenger RNA by hybridization with the mouse submaxillary gland (SMG) renin cDNA probe. The human kidney messenger RNA is about 1.6 kilobase (kb) long, similarly to the mouse SMG renin mRNA. A kidney renin cDNA clone of 1.1 kb length was obtained. A comparison of nucleotide sequences of mouse and human cDNA clones reveals conservation of residues involved in catalytic mechanisms and a potential glycosylation site. The human renin molecular probe allowed us to study renin expression in human chorionic tissue. The chorionic and kidney renin messenger RNAs are similar in length. The Southern blot analysis reveals the presence of a single renin gene in human DNA.     

Physicochemical properties of non-activated and activated renin from human amniotic fluid.

The main physicochemical and enzymic properties of non-activated and activated human amniotic renin (EC 3.4.99.19) were studied in order to clarify the relationships between the two enzymes. Human amniotic renin was activated by dialysis against acidic buffer (pH 3.3), direct acidification or trypsin treatment. All procedures produced similar activation. The physicochemical characteristics of non-activated and activated renin were compared to those of human renal renin. Non-activated renin had a molecular weight of 45,500. A similar molecular weight was obtained by gel eluate activation and by acid treatment of renin prior to gel filtration. Similar isoelectric points were also found for non-activated and activated renin. One major renin peak focused at pH 6.6, whereas no similar renin peak was detected in extracts from normal human kidney. In addition, non-activated and activated renin forms were found to have the same optimal pH, the same Km and the same inhibiting pepstatin concentrations.     

Mouse submaxillary renin has a protease activity and converts human plasma inactive prorenin to an active form

The activation of inactive prorenin by active renin was investigated. Inactive prorenin extensively purified from human plasma was activated by active renin which had been purified from mouse submaxillary glands by multiple chromatographic steps. The apparent lack of protease activity in renin was puzzling in view of the close similarity of its active site structure with that of acid proteases. After a series of affinity chromatographic steps designed to eliminate minute contaminants, renin was found to contain a very low but finite level of a neutral protease activity which was equivalent to 1/40,000 of that of cathepsin D tested by hemoglobinolytic activity. The protease activity was considered as intrinsic to renin since it co-purified with renin persistently at a constant ratio to the renin activity, was precipitated by a monoclonal antibody specific for renin, showed a neutral pH optimum of the enzyme activity in the same pH range as that of renin, and was inhibited by pepstatin. The neutral protease activity is likely to mediate the activation of inactive prorenin.     

The molecular biology of human renin and its gene

The molecular biology of renin, prorenin, and the renin gene have been studied. A tissue-specific pattern of expression was found in rat and human tissues. In the human placenta, the transfected and endogenous renin promoters are active, and renin mRNA levels and transfected promoter activity are increased by a calcium ionophore plus cAMP. Cultured pituitary AtT-20 cells transfected with a preprorenin expression vector mimick renal renin release by converting prorenin to renin and releasing renin in response to 8Br-cAMP. Studies with mutant renin genes suggest that the body of renin directs renin to the regulated secretory pathway, and renin glycosylation affects its trafficking. Chinese hamster ovary cells were used to produce recombinant prorenin. Infused prorenin was not converted to renin in monkeys. Renin crystals were used to determine its three-dimensional structure. Renin resembles other aspartyl proteases in the active site and core, but it differs in other regions that probably explain renin's unique substrate specificity. Based on structural and mutational analysis, a model for human prorenin was built that suggests lysine -2 of the prosegment interacts with active site aspartate residues, and that the prosegment inactivation of renin is stabilized by binding of an amino terminal beta strand into a groove on renin.     

Observations on the renal processing and sorting of prorenin.

Human prorenin is the biosynthetic precursor of renin. In general, prorenin is enzymatically inactive until it is converted to renin. The kidney is the major source of renin in the circulation, and is also an important source of circulating prorenin. The mechanisms of prorenin sorting and processing to renin in the juxtaglomerular cell may be a determinant of renal renin production. Therefore, our studies have focused on renal enzymes involved in "limited proteolysis" of prorenin to renin and on the morphology of prorenin sorting in the human juxtaglomerular cell.     

Purification of high molecular weight (HMW) renin from porcine kidney and direct evidence that the HMW renin is a complex of renin with renin binding protein (RnBP)

The high molecular weight (HMW) renin was purified from porcine kidney by a procedure involving extraction with a buffer system containing protease inhibitors, ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B column chromatography, gel filtration on Ultrogel AcA 44 and aminohexyl-Sepharose 4B column chromatography. The resulting preparation showed a single band on isoelectric focusing, exhibiting an isoelectric point at pH 5.25, and was stable on storage at -80 degrees C for 4 months. The specific activity was 3.97 mg of angiotensin I formed/mg of protein per h at 37 degrees C and at pH 6.5 with porcine angiotensinogen as the substrate. When the HMW renin was exposed to acid, renin activity increased by about 5-fold and the free form of fully active renin was recovered from the acidified HMW renin, leaving an insoluble aggregate of protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the HMW renin showed two protein bands, of which one was identified as renin from the electrophoretic mobility and the other was the protein, assigned as renin binding protein (RnBP), that was insolubilized by acidification. The purified HMW renin is a complex of renin with RnBP, and the molecular weights of RnBP and renin in the HMW renin were estimated to be 39,000 and 32,000, respectively, by gel permeation liquid chromatography in 6 M guanidine-HCl. A modified rapid method for purification of renin is also presented.     

The effect of thyroid hormone on renin production and release by rat kidney slices

The effect of thyroid hormone on renin productiona and release by rat kidney slices was studied. Rat kidney slices were incubated in Warburg flasks containing Krebs-Ringer-Phosphate- Glucose- Dextran solution at 37 C for 5 hours. Renin content, renin released into the incubation media and oxygen consumption were measured. Kidney slices actively secreted renin. Kidney slices of hyperthyroid rats released more renin, and kidney slices of hypothyroid rats released less renin than normal kidneys (p less than 0.001). The addition of 1-thyroxine to the incubation medium increased significantly (p less than 0.001) renin release by kidney slices from normal and hypothyroid rats. Thyroid hormone affects renin release through a mechanism independent of the ouabain-sensitive sodium pump and protein synthesis, since ouabain and cycloheximide did not modify renin release or production. The results of this study suggest that thyroid hormone plays a role in renin release from the juxtaglomerular cells.     

The human renin infused rat: use as an in vivo model for the biological evaluation of human renin inhibitors

The human renin infused rat model (HRIRM) was used as an in vivo small-animal model for evaluating the efficacy of a collection of inhibitors of human renin. The intravenous infusion of recombinant human renin (2.4 microg x kg(-1) x min(-1)) in the ganglion-blocked, nephrectomized rat produced a mean blood pressor response of 47+/-3 mm Hg (1 mm Hg = 133.3 Pa), which was reduced by captopril, enalkiren, and losartan in a dose-dependent manner following oral administration, with ED50 values of 0.3+/-0.1, 2.5+/-0.9, and 5.2+/-1.6 mg/kg, respectively. A series of peptidomimetic P2-P3 butanediamide renin inhibitors inhibited purified recombinant human renin in vitro in a concentration-dependent manner, with IC50 values ranging from 0.4 to 20 nM at pH 6.0, with a higher range of IC50 values (0.8-80 nM) observed at pH 7.4. Following i.v. administration of renin inhibitors, the pressor response to infused human renin in the HRIRM was inhibited in a dose-dependent manner, with ED50 values ranging from 4 to 600 microg/kg. The in vivo inhibition of human renin following i.v. administration in the rat correlated significantly better with the in vitro inhibition of human renin at pH 7.4 (r = 0.8) compared with pH 6.0 (r = 0.5). Oral administration of renin inhibitors also resulted in a dose-dependent inhibition of the pressor response to infused human renin, with ED50 values ranging from 0.4 to 6.0 mg/kg and the identification of six renin inhibitors with an oral potency of <1 mg/kg. The ED50 of renin inhibitors for inhibition of angiotensin I formation in vivo was highly correlated (r = 0.9) with the ED50 for inhibition of the pressor response. These results demonstrate the high potency, dose dependence, and availability following oral administration of the butanediamide series of renin inhibitors.     

Renin and hemodynamic changes via central adrenergic, cholinergic, and sodium receptor mechanisms in conscious rats.

The effects of centrally administered autonomic drugs and hypertonic saline on renin release were studied in the conscious rat. A 0.3 mug intraventricular dose of isoproterenol, which is one-thirtieth of the intraperitoneal dose required to stimulate renin release, induced the release of renin into the systemic circulation. Norepinephrine had no effect on renin release in the same dose range. Hypertonic saline and carbachol suppressed renin release. Alterations in renin release were preceded by a reciprocal change in blood pressure. These results suggest a central nervous system site for sodium, beta-adrenergic, and cholinergic receptors in altering renin release and blood pressure.     

The mouse Rn locus: S allele of the renin regulator gene results from a single structural gene duplication.

Inbred strains of mice have been divided into two distinct phenotypic groups having different levels of renin activity regulated by androgen in the submaxillary gland (SMG). Strains carrying the Rnrs allele of the renin gene regulator, located on chromosome 1, have a high level of renin activity; strains carrying the Rnrb allele have a low level of renin activity. The level of SMG renin activity correlates with the level of renin mRNA. We have analyzed, by Southern blot hybridization, the organization of renin genes in both strains. Strains carrying the Rnrb allele, such as BALB/c or C57 Bl/6, or CH3 mice, have one renin structural gene per haploid genome, while those having the Rnrs allele, such as AKR or Swiss mice, have two renin genes. We have also identified renin genes in mice belonging to different biochemical groups: Mus spretus has one renin gene while M. vrania and M. musculus brevirostris have two renin genes.     

Renin in the mouse submaxillary gland has a molecular weight of 40,000.

The availability of pure submaximillary renin, its antibody and pure specific immunoreactive Fab fragments of the antirenin molecule were used in an attempt to detect in which form renin is stored in the submaxillary gland. The proteolytic activity of serine-, metallo- and sulfhydryl enzymes during homogenisation was inhibited, but no inactive or high molecular weight form could be detected enzymatically or antigenically after gelfiltration. Nor were they demonstrable in crossed immuno-electrophoresis by using antibodies elicited against pure renin. Furthermore, pepstatin which additionally inhibits acid proteases, including a possible autoactivation of renin, and renin specific Fab fragments, were added, the latter in order to steric hinder proteolytic attack on a possible renin precursor. The renin-Fab complex was purified by precipitation with anti-Fab antibodies. No high molecular weight renin was demonstrable in SDS polyacrylamide gel electrophoresis. The only form of renin demonstrable in the submaxillary gland of mice was the fully active 40,000 dalton form. Its specific enzymatic activity was identical to that of pure submaxillary renin, being 0.4 . 10(-3) Goldblatt unit . ng-1.     

Ovarian renin gene expression is regulated by follicle-stimulating hormone

The regulation of renin and renin messenger RNA (mRNA) in the rat ovary was examined to test the hypothesis that the expression of renin gene and the secretion of renin in the ovary is the estrogen-mediated process that responds to follicle-stimulating hormone (FSH). In the ovary of the immature 25-day female rats, the concentration of renin mRNA was comparatively low, but 36 h after injection of FSH, the renin mRNA content showed a three-fold increase compared to the basal level. This increase was consistent with the stimulation of the total renin concentration in the ovary. On the other hand, the total renin concentration in the rat uterus gradually decreased, suggesting that the enhancement of the contents of renin and renin mRNA by FSH is an ovary-specific phenomenon. In hypophysectomized rats, the total renin concentration in the rat ovary was stimulated by the estrogen as well as FSH. These findings suggest that the production of ovarian renin is regulated by the pituitary hormone, particularly FSH.     

Identification of plasma inactive renin as prorenin with a site-directed antibody

A sequence-specific antibody that recognizes a portion of the prosegment of human renin precursor was raised and used to provide direct evidence that plasma inactive renin contains the prosequence of renal renin and is therefore probably prorenin rather than an inactivated form of previously active renin. The information may help not only to resolve a major controversy concerning the nature of inactive renin in human plasma but also to elucidate its exact physiological role.