Pure Human Renin Research Articles

Purification and characterization of human truncated prorenin.

Posttranslational processing of enzymatically inactive prorenin to an active form participates in the control of the activity of a key system involved in blood pressure regulation, growth, and other important functions. The issue is complicated because renin can be produced by a number of tissues throughout the body, in addition to the kidney, but the mechanism by which they process prorenin to renin is unknown and difficult to determine because of the small amounts of renin present. In the juxtaglomerular cell of the kidney, a 43 amino acid prosegment is cleaved from the amino terminus of prorenin to generate renin of molecular weight 44,000 [Do, Y. S., Shinagawa, T., Tam, H., Inagami, T., & Hsueh, W. A. (1987) J. Biol. Chem. 262, 1037-1043]. Using human uterine lining or a recombinant human prorenin system, we employed the same approach as that used in kidney, ammonium sulfate precipitation at pH 3.1 followed by pepstatin and H-77 affinity chromatography or gel filtration, to purify to homogeneity a 45,500-MW totally active renin. The specific activity of the active truncated prorenin was 850 Goldblatt units (GU)/mg of protein for chorion-decidua renin and 946 GU/mg of protein for recombinant renin, both similar to that reported for pure human renal renin. Both forms of renin cross-reacted with an antibody generated against 44,00-MW pure human renal renin and with an antibody generated against a peptide identical to the carboxy-terminal one-third of the prosegment.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for heterogeneity of glycosylation of human renin obtained by using lectins.

1. In this study, the carbohydrate structure of pure human renin was examined by using various lectins. 2. Pure renin could be separated into three forms by concanavalin A chromatography, a concanavalin A-unbound form, a loosely bound form and a tightly bound form, termed renins A, B and C, respectively. Renins A, B and C accounted for 3, 13 and 84%, respectively, of the purified renin. These forms were all present in individual human plasma and the relative proportions in plasma were 27 +/- 3, 33 +/- 4 and 39 +/- 5% (means +/- SEM) for renins A, B and C, respectively (n = 5). 3. Each form, electroblotted on to the nitrocellulose sheet after gel electrophoresis, was incubated with five peroxidase-labelled lectins, lentil lectin, erythroagglutinating phytohaemagglutinin, wheat-germ agglutinin, Ricinus communis agglutinin and peanut agglutinin. The protein was stained with 4-chloro-1-naphthol. 4. The staining pattern obtained with these lectins was significantly different among the three forms of human renin, confirming that they have different carbohydrate structures. Furthermore, the positive staining of human renin with erythroagglutinating phytohaemagglutinin, wheat-germ agglutinin and Ricinus communis agglutinin was in contrast with the lack of binding of rat renin to these lectins. 5. These results indicate the renal secretion of differently glycosylated multiple forms of human renin. The carbohydrate structure of human renin appears to differ from that of rat renin.

The distribution of renin‐containing cells in kidneys with renal artery stenosis—an immunocytochemical study

In 10 kidneys removed for hypertension due to renal artery stenosis, the histological appearances varied from negligible ischaemic damage to end-stage ischaemic atrophy. We stained the renin-containing cells in tissue sections using an antiserum to pure human renin and an immunoperoxidase technique. In all kidneys there was hyperplasia of the renin-containing cells both in juxtaglomerular apparatuses (JGAs) and in arteries outside the JGA, where these cells extended proximally as far as the interlobular arteries. We mapped the distribution of renin-containing cells and found them in all zones of the renal cortex; in three kidneys they were predominantly in the superficial cortex; in four they were distributed more evenly throughout the width of the cortex; but in three kidneys the normal gradient was reversed, with most of the cells being in the juxtamedullary cortex and, in two of the cases, little or no stainable renin in the superficial cortical JGAs. We suggest that these abnormalities in the distribution of renin-containing cells could affect both the pattern of intrarenal blood flow and the site in the kidney at which secreted renin enters the blood.

Immunological approach to blockade of the renin-substrate reaction.

To block the renin-substrate reaction by immunological tools is a long-standing dream. Since 1951 active immunization and the passive transfer of antirenin antisera have been successfully carried out in different species and in different experimental models of hypertension and normotension. These studies indicated that renin is a powerful immunogenic protein, capable of breaking down self-tolerance in different species. In this initial period the most significant results were obtained with hog renin. Passive transfer of antisera raised against hog renin or active immunization with hog renin was able to decrease blood pressure in renovascular or essential hypertension in dogs. Renin was semipurified and injected without adjuvant, however, since there was no method for determining plasma renin activity. Recently, complete purification of murine and human renin has allowed an extension of this approach, using the passive transfer of antirenin polyclonal antisera or monoclonal antibodies. Active immunization against pure human renin was successful in normotensive marmosets. This immunization with a nearly homologous renin in a primate model induced a significant decrease in blood pressure, associated with a complete disappearance of plasma renin activity. Unfortunately this powerful immunization was associated with an autoimmune disease that is specific for the kidney, related to self-recognition of the production site of renin by antibodies and lymphocytes. Similar results were reported with the use of mouse submandibular gland renin as an immunogen in spontaneously hypertensive rats (SHR). This manipulation decreased blood pressure in SHR to a level near that of normotensive Wistar-Kyoto control rats. However, again the animals showed a severe autoimmune disease of the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and Characterization of Renin-Producing Human Chorionic Cells in Culture

Chorionic tissue is one of the major extrarenal sites of renin production, and as such, cultured chorionic cells are a potential model for in vitro studies of renin biosynthesis and regulation. Human chorionic cells were isolated from four chorions and maintained in tissue culture for a total of eight subcultures. Total renin production was considerable in the primary cultures, but fell gradually with successive passages. The cells could be frozen and thawed without losing their ability to divide or produce renin. Both the primary cultures and the subcultures contained a single type of elongated cell containing abundant rough endoplasmic reticulum and myofibrils, but no renin granules, suggesting that the cells had smooth muscle-like features. Immunocytochemistry indicated that they contained both renin and prorenin. The renin produced by the chorionic cells was not stored within the cells, but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had biochemical and immunological properties similar to those of pure human renin. The cells contained a renin mRNA that had the same size as that for renal renin (1.6 kilobases), confirming the synthesis of renin by these cells. The cells were also examined for the presence of other components of the renin-angiotensin system. Angiotensinogen and angiotensin I were not detected, but angiotensin-converting enzyme was present in extracts of primary and secondary cultured cells. beta hCG and progesterone were also found in the medium of primary culture. However, the production of beta hCG and progesterone fell after the primary culture, and beta hCG and progesterone were indetectable in secondary and tertiary cultures, respectively. These experiments suggest that these two hormones do not influence renin synthesis or vice versa. Thus, these cultures of human chorionic cells synthesized considerable quantities of prorenin and can provide a permanent source of nonrenal prorenin-producing cells.

Renin in Glioblastoma Multiforme and Its Role in Neovascularization

Significant proliferation of capillaries with hyperplastic vascular endothelium is one of the characteristic histologic features of glioblastoma multiforme (GBM). It has been shown that the renin-angiotensin II cascade stimulates new vessel formation. The presence of renin in several types of highly vascularized neoplasm suggests that it may also be implicated in the mechanism of tumor angiogenesis. In order to study the possible relationship of renin to GBM, immunohistochemical search for human renin was carried out in ten instances of such a tumor. Eight of these cases demonstrated renin-containing neoplastic astrocytes, whereas seven cases of reactive gliosis and six cases of low-grade astrocytoma revealed no renin-containing cells. The immunostaining was not present after preabsorption of the renin antiserum with pure human renin or substitution of preimmune serum for the specific renin antiserum. Because it has also been demonstrated that a product of renin, angiotensin II, has angiogenic properties, it seems reasonable to postulate that renin, through angiotensin II, may play a role in the mechanism of GBM-associated neovascularization.

Characterization of five epitopes of human renin from a computer model

Antigenic determinants in proteins have been found to coincide with surface regions accessible to a large probe of 1-nm radius, which approximates the size of an antibody binding domain. To predict epitopes of human renin, a computer algorithm was used to calculate the accessibility of a model of the three-dimensional structure of human renin to a 1-nm probe. Of the 17 segments predicted to be epitopes or parts of epitopes, 6 were synthesized and tested for their recognition by 11 polyclonal antibodies prepared against pure human renin. Four peptides, Y-133-144, C-180-188, Y-211-224, and Y-300-310, were bound by some polyclonal anti-renin antibodies when tested in both a solution assay and an enzyme-linked immunosorbent assay (ELISA), and one peptide, C-290-296-G, was bound only in an ELISA. Of the five epitopes identified, peptide Y-211-224 seems dominant, since it was bound by all antisera in the solution assay and by 8 of the 11 in the ELISA. This peptide contains a disulfide loop, and the corresponding linear peptide obtained after reduction of the disulfide was not recognized by any anti-renin antisera in the solution assay. This suggests that the epitope is conformationally dependent and that a disulfide bridge linking cysteines-217 and -221 is present in the native structure of human renin. Purified immunoglobulins raised to peptides Y-133-144 and Y-211-224 (located near, but not at, the catalytic site) were found to inhibit the cleavage of human angiotensinogen by purified human renin but not the cleavage of a tetradecapeptide substrate representing the N-terminal region of angiotensinogen.(ABSTRACT TRUNCATED AT 250 WORDS)

Active immunization against renin in normotensive marmoset.

Primate renins (human and monkey) are very similar. We used pure human renin to immunize marmosets (Callithrix jacchus) and thereby produce a chronic blockade of the renin-angiotensinogen reaction. After a control period of 2 months, five male marmosets, on their usual sodium-poor diet, were immunized against pure human renin by three subcutaneous injections of 30 micrograms each, with complete and then incomplete Freund's adjuvant. Three marmosets were injected with adjuvant only and served as controls. Blood sampling and blood pressure measurements were performed weekly. After the third injection, the five marmosets immunized against renin developed a high titer of renin antibodies (50% binding of 125I-labeled human renin at a dilution of greater than or equal to 1:10,000). The antibodies inhibited the enzymatic activity of both marmoset and human renins. At the same time, systolic blood pressure decreased significantly from 125 +/- 13 mm Hg to 87 +/- 8 mm Hg (mean +/- SD; 1 mm Hg = 133 Pa). Plasma renin enzyme activity was undetectable in three animals. Plasma aldosterone decreased significantly. After 1-4 months with low blood pressure, a normal urinary output, and a normal plasma creatinine, the five marmosets became sick and died within one month. At autopsy an immunological renal disease, characterized by the presence of immunoglobulin and macrophage infiltration colocalized with renin, was found. Granulomatous formations, probably due to Freund's adjuvant, could be seen in the lungs and in the kidney. No immunoglobulin was detectable in extrarenal vessels or in other organs. These experiments demonstrate that, in this primate, a chronic blockade of the renin-angiotensin system can be achieved by active immunization against homologous renin, but this blockade is associated with the development of an autoimmune disease localized in the kidney.

Comparative enzymatic studies of human renin acting on pure natural or synthetic substrates

Some of the essential structural requirements for the enzymatic reaction of pure human renin acting on pure human and rat angiotensinogen and on their synthetic tetradecapeptide substrates were investigated. The five carboxy terminal amino acids of synthetic tetradecapeptides played a significant role in substrate recognition and/or hydrolysis by human renin. Kinetic constants K m, k cat and k cat/ K m of the various human renin assays were different according to the substrate used. The presence of either an asparagine or a threonine residue in the S′ 4 renin subsite did not affect significantly the kinetic constant values. A tyrosine residue, rather than a histidine residue, in the S′ 3 renin subsite gave the best synthetic substrate studied. When tyrosine residue was present in the S′ 2 renin subsite an improtant decrease in k cat was observed. Human angiotensinogen was hydrolysed by human renin with lower K m and k cat values than those measured with human and porcine synthetic substrates, suggesting that the 3-dimensional structure of human angiotensinogen plays a key role in the hydrolysis. This finding was supported by assays performed with rat angiotensinogen, which was cleaved by human renin with the same k cat value as rat tetradecapeptide, but with a 49-fold lower K m. Between human and rat angiotensinogen a k cat/ K m value of only 2-fold higher has been found in the renin assay using human substrate.

Production and characterization of human renin antibodies with region-oriented synthetic peptides.

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.

Characterization of pure human renal renin. Evidence for a subunit structure.

Renin was completely purified from human kidney cortex employing a rapid three-step procedure which included homogenization and ammonium sulfate precipitation, aminohexyl-pepstatin affinity chromatography, and affinity chromatography using a synthetic octapeptide renin inhibitor (H-77) with a reduced peptide bond (-CH2-NH- instead of -CO-NH-) between Leu5-Leu6, Three kg of cortex dissected from 10 kg of human cadaver kidney yielded 1.7 +/- 0.5 mg of protein (mean +/- S.E. for five procedures) with a specific activity of 1094 +/- 166 Goldblatt units/mg of protein and an overall recovery of 52 +/- 2%. Both gel filtration high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 44,000, although Mr = 22,000 and 18,000 bands were also identified by SDS-PAGE. The pH optima with sheep angiotensinogen were 5.5 and 7.8 and the Km was 0.31 microM. With pure human substrate the pH optimum was 6.0 and the Km was 1.15 microM. Enzyme activity was inhibited by two different anti-human renal renin antibodies. Amino-terminal sequencing demonstrated a leucine residue at the 1-position. Sequencing of 15 additional amino acids agreed with that predicted from the gene sequence and indicated that prorenin is converted to renin following cleavage at the carboxyl end of two basic residues, Lys-2 Arg-1. As with SDS-PAGE analysis, high performance liquid chromatography in the presence of 6 M urea demonstrated Mr = 44,000, 22,000, and 18,000 bands. Immunoblot studies revealed that all of these bands cross-reacted with antihuman renin antibody. Amino-terminal sequencing indicated the Mm = 22,000 band is the amino terminus and the Mr = 18,000 band the carboxyl terminus of Mr = 44,000 renin. In the aqueous phase, these subunits bound to H-77 suggesting that they represent components of the active enzyme complex. Unlike mouse renin, there was no evidence of disulfide bonds. These results raise the question of whether human renin circulates as a subunit aggregation as well as a single chain protein. This may serve as a possible mechanism to regulate renin activity in plasma and tissues.

Study of the antigenic determinants of human renin.

The primary structure of human renin, recently established from the complementary DNA sequence of its messenger RNA, shows a strong homology to other aspartyl proteases. This homology has permitted the construction of a model of the three-dimensional structure of renin based on the crystallographically determined structures of three aspartyl proteases: penicillopepsin, endothiapepsin, and rhizopuspepsin. Using an algorithm in which a spherical probe approximating the size of the antibody-binding domain (1-nm radius) was allowed to contact the surface of the renin model, we predicted 12 to 15 peptides to be immunogenic epitopes. We synthesized peptides corresponding to three different regions of the model: Cys-Gly-Ser-Asp-Pro-Gln-His-Tyr-Glu-Gly-amide (C-180-188), Tyr-Leu-Leu-Cys-Glu-Asp-Gly-Cys-Leu-Ala-Leu-amide (Y-215-224; disulfide bond between cysteines) and Tyr-Gly-Ser-Ser-Thr-Leu-Leu-Cys-Glu-Asp-Gly-Cys-Leu-Ala-Leu-amide (Y-211-224; disulfide bond between cysteines), and Cys-Tyr-Ser-Ser-Lys-Lys-Leu-Cys-Gly (C-290-296-G; disulfide bond between cysteines). All four peptides were tested for their binding to 11 polyclonal and 7 monoclonal antibodies raised against pure human renin, in both a solution assay and an enzyme-linked immunosorbent assay. Peptides Y-215-224 and Y-211-224 bound to all 11 polyclonal antibodies in the solution assay, and peptide Y211-224 bound to eight of them in the enzyme-linked immunosorbent assay. Therefore, region 211-224 can be identified as a major epitope of the human renin molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of renin-producing human cells by transfection with three simian virus 40 mutants.

A human juxtaglomerular cell (JGC) tumor was used for the immortalization of renin-secreting cells. The transfection of primary JGC with three different simian virus 40 (SV40) mutants resulted in the continuous production of renin-secreting cells. The most efficient renin-producing cells (producing about 400 pg of renin per 24 hr per ml of culture medium) were those transfected with the PAS SV40 mutant. The renin production was stable and the cell cultures have been maintained for greater than 1 year. Two types of cells were cultured together and could not be separated: round and birefringent cells, which exhibited features of mast cells, and elongated cells containing myofilaments and secretory granules. Immunocytochemical staining showed the presence of renin in this latter cell type. The renin produced by the transfected cells was not stored within the cells but was released rapidly into the medium. More than 95% of the renin produced was prorenin, which, after activation, had characteristics similar to those of pure human standard renin as to its enzymatic, immunologic, and biochemical properties, except that it was less glycosylated. These stable JGC tumoral cell lines provide a unique system for studying human renin biosynthesis and its regulation in vitro.

The granular peripolar cell of the human glomerulus: a new component of the juxtaglomerular apparatus?

Using serial sections of resin-embedded tissue we found granular peripolar cells in six human kidneys. They were present in 3% to 28% of the glomeruli. Using an immunoperoxidase staining technique and an antibody to pure human renin we showed that the human peripolar cell contains no immunostainable renin. The number of peripolar cells correlated with the number of juxtaglomerular apparatus (JGAs) with renin-containing cells; their distribution within the renal cortex was similar, both being found predominantly in glomeruli in the superficial cortex. There was a close anatomical relationship between the peripolar cells and the renin containing cells in individual JGAs. These findings suggest the possibility of a functional relationship between the peripolar cell and the other components of the juxtaglomerular apparatus.

Erythropoietin and Renin Substrate in Cerebellar Haemangioblastoma

We examined eight cerebellar haemangioblastoma tumours from eight patients, aged 16-63 years, 5 females and 3 males. Preoperative haemoglobin values exceeded 180 g/l in four patients, and 150 g/l in four. All high Hb values were normalized upon surgical removal of the tumours. All tumours contained scattered cells which stained positively with antisera against pure human urinary erythropoietin and plasma renin substrate. We conclude that cerebellar haemangioblastomas produce immunoreactive erythropoietin, which shares common antigenic determinants with renin substrate.

Immunocytochemical localisation of renin in nephroblastoma.

An antibody to pure human renin and an immunoperoxidase technique were used to stain immunoreactive renin in nephroblastomas. In an unselected series of surgically removed nephroblastomas immunoreactive renin was found in 10 of 19 tumours (53%). Tissue obtained at necropsy from 12 cases of disseminated nephroblastoma was also studied, and immunoreactive renin in metastatic tumours was found in three cases. Patients' blood pressures and serum potassium concentrations showed no evidence of excess renin activity. It is therefore suggested that if nephroblastomas contain renin as commonly as this study suggests, then any hormone secreted may be biologically inactive.

Blood pressure effects of renin inhibition by human renin antiserum in normotensive marmosets.

The effects on blood pressure of an antiserum against pure human kidney renin were studied in conscious and anesthetized (pentobarbital, 24 mg X kg-1 i.p.) small new world monkeys (common marmosets). The antiserum inhibited the enzymatic activity of renin by 50% in a dilution of 1:45,000 in marmoset and 1:50,000 in human plasma. The antiserum (0.2 ml i.v.) decreased blood pressure in conscious marmosets on normal sodium intake by 15 +/- 5 (SD) mmHg and after salt depletion by 31 +/- 13 mmHg. A converting enzyme inhibitor (teprotide, 2 mg X kg-1 i.v.) induced a comparable fall in blood pressure: -16 +/- 10 and -30 +/- 10 mmHg, respectively. Similar effects were observed on blood pressure of anesthetized marmosets. The correlation between pretreatment plasma renin concentration and the maximum fall in blood pressure was significant and identical for the experiments with antiserum and teprotide. These results demonstrate that antisera against human renin can be used for the specific blockade of the renin-angiotensin system in primates. In normotensive marmosets the renin-angiotensin system participates in the maintenance of blood pressure, to a degree depending on the state of sodium balance.

New morphological evidence for the synthesis and storage of renin in the human kidney: an ultrastructural immunocytochemical study.

Using an antibody to pure human renin and the technique of ultrastructural immunocytochemistry we have localized renin in the granular epithelioid cells of three juxtaglomerular apparatuses and one interlobular artery. The staining reaction of the cells was the same in both sites: we found that crystalline protogranules, rounded membrane-bound granules and storage granules intermediate in morphology all stained positively for renin. We also demonstrated immunoreactive renin in dilated cisternae of rough endoplasmic reticulum in the granular cells in both sites. We present these observations as morphological evidence that granular epithelioid cells synthesize and store renin both in the juxtaglomerular apparatus and in the larger arteries in the kidney.

Antisera to human renin normalize renin-dependent hypertension in the monkey: therapeutic implications.

Polyclonal antisera raised against pure human renin normalize renin-dependent blood pressure elevation in the monkey (M. fascicularis). In vitro, comparable inhibition of either human or monkey plasma renin by the antisera was demonstrated. In vivo, intravenous infusion of 2 ml of antisera did not change mean arterial pressure of salt-repleted monkeys, however, its administration to salt-depleted monkeys with elevated plasma renin activity lowered mean arterial pressure 10 Torr. A 25 Torr rise in mean arterial pressure and increase in plasma renin activity occurred promptly after inflation of a suprarenal aortic cuff in conscious uninephrectomized monkeys. Administration of 2 ml of antisera to these monkeys normalized mean pressure, which was reduced by an additional 10 Torr if the animals were previously salt-depleted. Maximal hypotension occurred within 1 hour and was sustained for approximately 10 hours. Because of the differing specificities of polyclonal antisera, sera raised in two laboratories against human renin purified from different sources were employed. Identical results were obtained. This is the first demonstration of the use of antisera to inhibit endogenous renin activity in primates and predicts the in vivo efficacy of renin antisera as experimental, diagnostic and therapeutic agents.

Renin in renal cell carcinoma--an immunocytochemical study using an antibody to pure human renin.

We have studied a series of 19 primary renal cell carcinomas using an antibody to pure human renin and the indirect PAP technique. Seven tumours contained immunoreactive renin. No renin was identified in seven cases of metastatic tumour within the kidney, but immunoreactive renin was present in two out of seven metastatic renal cell carcinomas in other organs. None of the subjects had clinical, biochemical or histological evidence of excess renin secretion. We suggest that renal cell carcinoma may commonly secrete renin and that the hormone may be biologically inactive.