Enzymatically inactive human renin from chorionic cells in culture is significantly distinct in polyacrylamide gel electrophoresis (pH 8.17, 0 degree C) from active human kidney renin. The inactive renin is larger and more basic than the active renin; their molecular weights derived from gel electrophoretic retardation coefficients relate as 47.5/35.3 kDa, their valences (net protons/molecule) as 2.14/1.85. In gel electrofocusing conducted in a mixture of simple buffers, both inactive and active renins exhibit 2 components at the steady-state. The molecular size and basicity of inactive renin are consistent with the hypothesis that it may be a precursor (prorenin), although the possibility that it is an inhibitor complex cannot be ruled out.

Monoclonal antibodies directed against human renin were obtained by the fusing of myeloma cells with spleen cells from Balb/c or high-responder Biozzi mice injected with pure tumoral or highly purified renal renin. These procedures resulted in the production of seven stable monoclonal antibodies to human renin. Antibodies in the hybridoma culture medium were screened by binding to pure iodinated renin or insolubilized renin in a solid phase assay. The concentration of purified antibodies that provided a 50% binding to iodinated renin varied from 1 X 10(-10) to 1 X 10(-7) M. Two monoclonal antibodies were found to be potent inhibitors of renin enzymatic activity in vitro, behaving as noncompetitive inhibitors (Ki, 1 to 4 X 10(-10) M). They were specific for primate renin. Three monoclonal antibodies provided suitable immunoadsorbants for renin purification. One of these immunoadsorbants was used for large-scale purification of the renal enzyme, resulting in an 825-fold renin enrichment in a single step. Two antibodies were able to distinguish between active and inactive renin and enabled concomitant separation and purification of the two enzyme forms in various biological fluids. Monoclonal antibodies also stained human and monkey renal renin when indirect immunofluorescence and peroxidase-antiperoxidase techniques were used. A highly sensitive radioimmunometric assay of renin was constructed with two monoclonal antibodies. The sensitivity of this improved assay should permit the detection of renin in normal human plasma. Monoclonal antibodies have been shown to be superior to polyclonal antibodies in the following areas: the separation of active from inactive renin, the purification of renin from biological fluids, and the setting up of a direct assay of plasma renin.

Renin biosynthesis was studied in a juxtaglomerular cell tumor. The tumoral tissue had a high renin content (180 Goldblatt Units/g of tissue), was heavily stained by immunofluorescence using human renin antiserum, and exhibited numerous characteristic secretory granules by electron microscopy. In one series of experiments, renin biosynthesis was studied in tissue slices, by following the incorporation of radiolabeled amino acids into specific immunoprecipitable renin. Time course studies showed that renin was first synthesized in a high molecular weight form, 55,000 mol wt, i.e., 10,000 mol wt higher than that of active renin, and was then converted into a 44,000-mol wt form. In a second series of experiments renin tumoral cells were cultured. Small, round, birefringent cells obtained after collagenase digestion produced renin in both primary culture and subculture media. After 5 d most of the renin found in the culture medium was inactive, but could be activated by trypsin treatment. The tumoral tissue exhibited a strong renin immunofluorescence and numerous secretory granules were observed by electron microscopy. In contrast, the renin-producing cells isolated from this tumor and grown in culture showed little renin immunofluorescence and no secretory granule could be observed. The renin-producing cells in primary culture and subculture were pulsed with radiolabeled amino acids, and immunoprecipitable radiolabeled renin was found in the culture media, thus demonstrating the actual biosynthesis of the enzyme. This renin was not stored inside cultured cells but was rapidly released into the medium and had a molecular weight of 55,000. No conversion of this inactive high molecular weight renin into the active, 44,000 mol wt form of renin was observed. We postulate the existence of two pathways for the processing, packaging, and secretion of renin in the tumoral cells: in juxtaglomerular cells of tumoral tissue renin is synthesized as a preprorenin and rapidly converted into prorenin (55,000 mol wt), which is in turn packaged in secretory granules where it is processed into active renin (44,000 mol wt) and finally secreted; in the cultured tumoral cells renin is still biosynthesized as a preprorenin molecule and then converted into prorenin, but is neither stored as granules nor processed into active renin. In this case the renin is released in an inactive form.

Amnion and chorion cells from human fetal membranes have been cultured. Chorionic cells secrete renin whereas amnionic cells do not. Renin is secreted by chorionic cells as an inactive form that can be activated by trypsin treatment or acid dialysis. The antigenic and enzymatic properties of activated chorionic renin and kidney renin are similar. Incorporation of [35S]methionine in the culture demonstrates that chorionic renin is secreted as a high molecular weight form, 54K. This 54K inactive renin could represent the proenzyme.

Primary cultures of human chorionic cells have been developed. They secrete an inactive form of renin which can be activated by trypsin treatment. In biosynthesis experiments forming labelled renin the inactive renin exhibited a molecular weight (54 KD) higher than active kidney renin (44 KD). The same high molecular weight renin was obtained during biosynthesis experiments performed on tissue slices of human infarcted kidney. Therefore it is postulated that 54 KD biosynthetized renin might be the precursor of active renin.

Biochemical and immunological characteristics of renin secreted by two malignant renin-secreting tumors [pulmonary (PT) and paraovarian (POT)] were studied. They both contain inactive renin (IR), as renin activity of tumoral extracts was able to be increased after acid activation or trypsin treatment (10.1 to 20.8 Goldblatt units/g tissue for PT and 1.4 to 3.71 for POT). Renin activity after activation reached the value obtained by direct RIA of human renin (23 and 3.4, respectively), as both forms are recognized by renin antiserum. Both enzymatic activities could be completely inhibited by renin antiserum. Displacement curves for the two tumoral renins paralleled the MRC renin in the direct RIA. After chromatography on affigel blue, active renin was not bound to the gel, and inactive renin eluted only with 1 M NaCl. On pepstatin A Sepharose and CBL-pepstatin Sepharose (an N-modified-pepstatin), a separation of the two forms of pulmonary renin was obtained; inactive renin eluted with breakthrough proteins, whereas active renin was strongly bound to the gel. After this affinity chromatography, the molecular weights of inactive and active renin, determined on Ultrogel, were very close (46,000 and 42,500). We conclude that 1) ectopic renin in these cases in similar to the renal enzyme; 2) renin can be secreted in an inactive form, supporting the hypothesis of an inactive initial state of renin; and 3) molecular weight differences between the two forms are very slight.

1. A mouse hybridoma secreting an antibody directed against human renin was obtained by fusion of spleen cells with NS 1 myeloma cells. This monoclonal antibody recognizes human and monkey renins but neither hog nor mouse renins nor the acid proteinase cathepsin D. 2. Monoclonal antibody was coupled to Sepharose 4 B. The immunoadsorbent thus obtained was very efficient in purifying both active and inactive human renin from renal or extrarenal sources. 3. Renin enrichment up to 3500-fold was obtained in a single step.

By spleen cell fusion with NS1 myeloma, a mouse hybridoma was obtained which secretes an antibody directed against human renin. This monoclonal antibody recognizes human and monkey renin, but neither hog nor mouse. Preliminary experiments demonstrate the potential of this antibody for renin immunopurification and characterization.

The first complete purification of renin (EC 3.4.99.19) has only been achieved during this last decade. The instability of the unpurified enzyme and its extremely low concentration in kidney and biologic fluids explain the difficulties encountered during the course of the purification. The complete purification of renin is a mandatory step for our understanding of the renin-angiotensin system, allowing the production of highly specific renin antibodies and the study of the possible renin precursors, the active site of the enzyme, its structure, and the mechanisms of its inactivation.

Human plasma and amniotic liquid were activated by dialysis at pH 3.3. Then, renin before and after acidification was determined by two methods: enzymatic activity measurement, and direct radioimmunoassay. The identity between nonactivated and activated renin in plasma and amniotic fluid on the one hand, and pure renin on the other, was demonstrated by the dilution curves in radioimmunoassay. After acidification, mean plasma renin activity in 17 patients with high renin activity rose from 26.8 +/- 11.7 pmoles A I ml-1 h-1 to 67.9 +/- 29.3 pmoles A I ml-1 h-1, whereas the mean renin concentration tested by direct radioimmunoassay remained constant at 13.8 +/- 10.5 and 14.8 +/- 11.2 fmol/ml before and after acidification respectively. In amniotic fluid, renin activity increased from 9.7 to 227 pmoles angiotensin I/ml/h, but the renin concentration did not change. Direct radioimmunoassay of renin may therefore be considered as measuring total renin, regardless of its enzymatic activity. In 12 hypertensive patients undergoing bilateral renal-vein catheterization, the direct measurement of renin was very significantly correlated to the non-activated (r = 0.883) and activated renin values (r = 0.963).

1. Human plasma, amniotic fluid and acidified amniotic fluid were incubated at pH 5.5 with the same concentrations of human plasma renin substrate and rat plasma renin substrate. They produced three to eight times more angiotensin I with human than with rat renin substrate. By contrast, human brain extracts generated 20 times more angiotensin I when incubated with rat plasma renin substrate than with human plasma renin substrate. 2. Serial dilutions of anti-(human renin) antibody inhibited, in a dose-dependent manner, the production of angiotension I when plasma, amniotic fluid and brain extracts were incubated with human plasma renin substrate. They also inhibited the production of angiotensin I when plasma and amniotic fluid were incubated with rat plasma renin substrate. They were ineffective on the angiotensin I generation by human brain extracts acting on rat plasma renin substrate. 3. Affinity chromatography on an haemoglobin-Sepharose gel separated the fraction of brain extract acting on human renin substrate and inhibited by anti-(human renin) antiserum; this was not retained on the gel at pH 3.3. Part of the angiotensin I-forming activity detected by rat renin substrate hydrolysis was not retained on the gel and part was eluted at pH 8.5. These angiotensin I-forming activities did not hydrolyse human renin substrate, and were not neutralized by anti-(human renin) antibody. 4. These results demonstrate that a renin, immunochemically identical with renal, plasma amd amniotic fluid renin, is present in the human brain. Other angiotensin I-forming activity, acting on an heterologous substrate at a more acidic pH, is also present in human brain.

Antibodies were raised in rabbit against pure human renin. The antisera obtained are highly specific for human renin versus hog, dog and rat renin. They do not cross-react with acid proteases such as pepsin and human cathepsin D. A direct radioimunoassay is described for human renin in plasma and kidney extracts. 30 to 50 pg of enzyme (2.5 to 4 x 10(-5) Goldblatt units) are detected.

Human renin was purified from a juxtaglomerular cell tumor with a high renin content, 24.2 Goldblatt units/mg of protein. The purification procedure comprised three steps: gel filtration, DEAE-cellulose chromatography, and preparative isoelectric focusing. Five forms of renin amounting to 5.3 mg of enzyme were obtained with isoelectric points of 4.95, 5.10, 5.35, 5.55, and 5.70. They were all glycoproteins. The three major fractions had very similar specific activities, 868, 860, and 809 Goldblatt units/mg of protein. These fractions produced a single band on analytical isoelectric focusing and a single arc on immunoelectrophoresis. On polyacrylamide gel electrophoresis at pH 7.8, each fraction consisted of two renin bands with the same molecular weight, but different net charges. The molecular weight determined by gel filtration and Fergusson plot analysis on polyacrylamide gel was 38,000 to 42,000. The optimum pH determined on N-acetyltetradecapeptide substrate was 6.5, and the K,,, was 6.8 x low6 M. These parameters were identical with those for standard human kidney renin. Antibodies raised against tumor renin completely inhibited the activity of both tumor and standard renin. Under dissociating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel electrophoresis in the presence of 6 M urea), part of the purified enzyme dissociated into two smaller fragments (I& = 20,000 and 25,000) containing renin activity.

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.

Human renin was purified from a juxtaglomerular cell tumor with a high renin content, 24.2 Goldblatt units/mg of protein. The purification procedure comprised three steps: gel filtration, DEAE-cellulose chromatography, and preparative isoelectric focusing. Five forms of renin amounting to 5.3 mg of enzyme were obtained with isoelectric points of 4.95, 5.10, 5.35, 5.55, and 5.70. They were all glycoproteins. The three major fractions had very similar specific activities, 868, 860, and 809 Goldblatt units/mg of protein. These fractions produced a single band on analytical isoelectric focusing and a single arc on immunoelectrophoresis. On polyacrylamide gel electrophoresis at pH 7.8, each fraction consisted of two renin bands with the same molecular weight, but different net charges. The molecular weight determined by gel filtration and Fergusson plot analysis on polyacrylamide gel was 38,000 to 42,000. The optimum pH determined on N-acetyltetradecapeptide substrate was 6.5, and the Km was 6.8 x 10(-6) M. These parameters were identical with those for standard human kidney renin. Antibodies raised against tumor renin completely inhibited the activity of both tumor and standard renin. Under dissociating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel electrophoresis in the presence of 6 M urea), part of the purified enzyme dissociated into two smaller fragments (Mr = 20,000 and 25,000) containing renin activity.

A simple fluorimetric assay was set up to test renin within 2 h. N-acetyltetradecapeptide was synthesized and used as substrate. It was demonstrated that N-acetyl-angiotensin I and Leu-Val-Tyr-Ser were the two peptides obtained after hydrolysis by renin. Fluorescamine reacted with the free NH2 of the tetrapeptide generated to induce a fluorimetric reaction detected at 395–495 nm. The Michaelis constant of the reaction was 1.87 · 10−5 M. With this method as little as one milliGoldblatt Unit (mG.U.) of hog renin could be detected and the generation of tetrapeptide was linear with respect to the renin concentration up to 20 mG.U. The fluorimetric assay was applied to the detection of renin during its purification and to the characterization of renin inhibitors.

Renin was purified from 47 kg of hog kidney to produce enough enzyme for enzymatic and physicochemical characterization. The procedure included extraction at pH 3.5 in the presence of protease inhibitors, two ammonium sulfate precipitations, ion exchange chromatography on Sepharose-hexamethylenediamino-pepstatin gel, gel filtration, and isoelectric focusing. Renin, 2.3 mg, with a specific activity of 1,100 GU/mg of protein was obtained with about 70,000-fold purification and 16% overall recovery. The purity criteria were: (1) a single band on sodium dodecyl sulfate (SDS)-gel electrophoresis, (2) same retardation factor on polyacrylamide gel electrophoresis for renin activity, protein and glycoprotein coloration. Renin was characterized by its stability at--20 degrees C, pH 6.5; its molecular weight on SDS-gel electrophoresis, 36,800; its relative mobility on polyacrylamide gel electrophoresis at pH 7.8; its isoelectric point, 5.15; its amino acid composition, which revealed that renin is a glycoprotein; and its Michaelis constant on tetradecapeptide substrate at pH 6.6, Km = 7.7 X 10(-6) M.