Purification of canine plasma renin by affinity chromatography.

An affinity column for the purfication of canine plasma renin was prepared using goat anti-renin (dog kidney) gammaG gloublins. The antiserum was prepared against a purified kidney renin preparation. The anti-renin globulins were coupled to cyanogen bromide activated Sepharose. Using the anti-renin globulin-coupled Sepharose as an immuno-adsorbant, a method was devised allowing purification of plasma renin to a 1,000-fold purity.     

Purification and characterization of rat urinary renin

Rat urinary renin was purified by a procedure involving ammonium sulfate fractionation, pepstatin-aminohexyl-Sepharose 4B chromatography, ion exchange chromatography and gel filtration. The resulting preparation was essentially homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 39000 by SDS-gel electrophoresis and 40000 by gel filtration. The optimum pH determined with rat angiotensinogen was 7.0, and the Km was 3.6 microM. These properties agreed well with those of purified rat renal renin. The activity of urinary renin was specifically inhibited by anti-renin antibody. These results suggest that urinary renin may originate in the kidney.     

Large scale preparation of pure hog kidney renin

The complete purification of renin raises difficult problems due to its extremely low concentration in kidney (less than 1/50,000 of total proteins). The complete purification of hog kidney renin has been realized on a large scale, starting from 300 kg of fresh hog kidneys. 14.6 mg of pure renin were obtained with an overall yield of 4%. The purification procedure involved 14 steps. The enzyme was extracted at pH 3.5. Subsequent purification steps were performed in the presence of protease inhibitors to decrease renin proteolysis. These steps included an ammonium sulfate precipitation and a batch-chromatography on DEAE-cellulose. The major purification step was an affinity chromatography on Sepharose-hexamethylene-diaminopepstatin. The enzyme obtained was further purified by molecular sieving gel filtration and isoelectric focusing.     

Purification of human renin by affinity chromatography using a new peptide inhibitor of renin, H.77 (D-His-Pro-Phe-His-LeuR-Leu-Val-Tyr).

A new affinity column for renin was prepared by coupling the isosteric peptide inhibitor of renin, H.77 (D-His-Pro-Phe-His-LeuR-Leu-Val-Tyr, where R is a reduced isosteric bond, -CH2-NH-), to activated 6-aminohexanoic acid-Sepharose 4B. Chromatography of a crude extract of human kidney cortex on this material resulted in a 5500-fold purification of renin in 76% yield. The purified enzyme (specific activity 871 units/mg) was free of non-specific acid-proteinase activity and was stable at pH 6.8 and -20 degrees C over a period of several weeks.     

Purification and characterization of human kidney renin

By means of a new rapid and small scale purification method, human kidney renin has been purified from a single kidney in a homogeneous state, as judged on SDS-PAGE. The kidney which showed unusually high renin activity was from a patient with cardiomyopathy. 8,000-fold purification was attained by means of only pepstatin-aminohexyl-Sepharose chromatography and FPLC on a Mono Q column, and the yield was 34%. The specific activity was 5.63 mg angiotensin I per mg protein per h at 37 degrees C and pH 6.5 with porcine angiotensinogen as the substrate. The molecular weight was estimated to be 37,000 by SDS-PAGE and 38,000 by HPLC on a TSK G-3000 SW column. The preparation showed three bands on isoelectric focusing. The molecular weight and the profile on isoelectric focusing of the purified renin agreed with those found for the extracts of both the patient's kidney and a kidney with the usual low renin activity.     

Isolation of pure and stable renin from hog kidney

Pure renin from hog kidney was isolated as an electrophoretically homogeneous preparation. An affinity column suitable for this purpose was prepared by soupling pepstatin to aminohexyl agarose in an organic solvent mixture. Crude extract, treated with mixtures of protease inactivators to eliminate proteases, was purified on this column followed by gel filtration and ion-exchange chromatography. Hog renin, thus obtained after 180,000-fold purification at an overall yield of 25%, is stable at pH6.35 and ?20°.     

Biochemical evidence for existence of immunoreactive renin in human prolactinoma tissue

High activity of renin was demonstrated in human prolactinoma tissue. This activity was almost completely inhibited by specific antibody raised against human renal renin, indicating that it was not due to the nonspecific action of proteases. The specific activity of renin was 5.04 ng of angiotensin I generated/mg of protein per h, comparable to that of the pituitary tissue prepared from postmortem human subjects. The biochemical properties of the prolactinoma renin were generally similar to those of well-known kidney enzyme, such as molecular mass (Mr = 46,000), optimum pH (6.0), and glycoprotein nature. However, the isoelectric points (pI) of the prolactinoma renin (pI = 4.90, 5.04, 5.24 and 5.41) differed somewhat from those of plasma and kidney renins reported hitherto. These results indicate that true renin can be produced in human prolactinoma tissue.     

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)     

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments.     

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.     

Blockade of AT1 receptors by losartan did not affect renin gene expression in kidney medulla

This study was designed to determine particular changes in the renin gene expression and activity in renal cortex and medulla after AT(1) receptor blockade. It was found that two-week-treatment with AT(1) blocker losartan induced an increase in tissue renin activity in both parts of kidney causing subsequent elevation of plasma renin activity. Renin mRNA in losartan-treated rats was increased only in cortex, suggesting cortex origin of elevated renin activity in medulla. Medullary renin mRNA indicated local synthesis of renin within the whole kidney and supported the idea of the presence of tissue renin-angiotensin system. Our results show that gene expression of renin in kidney medulla is insensitive to AT(1) receptor blockade and this points out that the regulation of kidney renin-angiotensin system probably differs from that in cortex.     

Purification of human plasma inactive renin by immunoaffinity chromatography on profragment-specific IgG

Inactive renin was purified to apparent homogeneity from human plasma by ion exchange, gel filtration, Affi-Gel blue, immunoaffinity chromatography on profragment-specific IgG coupled to Sepharose, and preparative HPLC. By this method, a 460000-fold purification was obtained. The purified renin was totally inactive and was activated by trypsin.     

Multiple defects in the renin-angiotensin system in alloxan-diabetic kidney

A defect in the renin-angiotensin system has been shown in diabetic patients and experimental animals, in particular with nephropathy or autonomic neuropathy. The mechanism for this low plasma renin activity (PRA) is poorly understood. In order to clarify this defect, the renin-angiotensin system was studied in alloxan-induced diabetic and age-match control mice. In diabetic animals, kidney renin activity (KRA) was significantly lower than that of the controls, while plasma renin substrate (PRS) concentration was slightly higher and PRA was normal. The amount of injected radiolabeled renin extracted by the kidney was normal, but the amount extracted by the liver was significantly decreased in diabetic animals. On the other hand, the degradation of the extracted renin by both the kidney and the liver was elevated as compared to the controls. This high degradation rate was accompanied by a slight increase in lysosomal protease activity in the kidneys. In in vivo studies, isoproterenol-induced PRA was 20-fold in control animals. In diabetics, isoproterenol-induced PRA was attenuated and rose only four- to fivefold over basal level. The angiotensin converting enzyme (ACE) activity in the kidney was significantly decreased in the diabetic state. It is concluded that there were multiple defects in the renin-angiotensin system in this diabetic model, namely, a depletion of renin storage with subsequent loss of maximal responsiveness to the adrenergic agonist in renin release, an elevation of intrarenal renin degradation together with a deficiency in ACE which would possibly lead to a decrease in intrarenal formation of angiotensin II.     

In vitro evidence for a blood-borne renin-releasing factor

The present studies using kidney slices were designed to test whether serotonergic stimulation of renin secretion is mediated via an endocrine signal. Previous in vivo studies have indicated that central serotonergic neurons regulate renin secretion. Administration of the serotonin releaser dl-p-chloroamphetamine-HCl (PCA) to rats causes dose-dependent increases in renin secretion that can be blocked by serotonin depletion with p-chlorophenylalanine (PCPA), injections of 5,7-dihydroxytryptamine into the dorsal raphe nucleus or ablation of the mediobasal hypothalamus. The renin-releasing substance was obtained from nephrectomized male donor rats which were sacrificed 1 hour after receiving an injection of PCA intraperitoneally. Plasma from rats that received saline injections was used as control. The plasma was collected and separated by ultrafiltration into fractions containing solutes with molecular weights between 500-10,000 daltons. The renin-releasing ability of this substance was studied in vitro using rat renal cortical slices. The plasma fraction (M.W. = 500 - 10,000) from rats treated with PCA caused dose-dependent increases in renin release from the kidney slices. Heating of the plasma factor at 100 degrees C for 30 minutes did not reduce the ability of this substance to release renin from the kidney slices. PCA alone (66 X 10(-6)M) did not increase renin release from the kidney slices. These data suggest that stimulation of serotonergic receptors in the brain triggers the release of an endocrine factor that is capable of directly stimulating renin release from the kidneys.     

Purification of rat renal renin from crude kidney extracts by diaminohexamethylene-Sepharose chromatography

Renin from rat kidney extracts was adsorbed to diaminohexamethylene-sepharose columns at extremely low ionic strength and neutral pH. Renin was retarded while the column was developed in 1 mM sodiumpyrophosphate and extraneous proteins were removed. Elution of renin was performed using a linear gradient of sodiumpyrophosphate, 1 – 17 mM at pH 6.8. Renin was purified in a yield up to approx. 60 per cent of the applied activity and a purification factor between 5 – 122 depending on the specific activity of the applied sample. The specific activity after this single chromatography of crude rat kidney homogenate on diaminohexamethylene-sepharose showed a median of 11.3 Goldblatt units per mg protein in a range of 5.3 – 42.0 Goldblatt units per mg protein. The renin binding capacity of the column was 1 Goldblatt unit per ml wet gel. The purified renin was subjected to G-100 Sephadex chromatography demonstrating two molecular weight forms of 44000 and 50000 dalton. Polyacrylamide gel electrophoresis demonstrated three separate fractions of renin.     

Renin activity determination using human plasma as a substrate

A method for human renin activity determination using human plasma as the substrate is described. Angiotensin I is generated by incubating renin with human plasma, which is available along with the commercial radioimmunoassay kit for angiotensin I. The method obviates the need to isolate and purify the substrate, human angiotensinogen, from human plasma. In addition, the assay is highly renin specific, sensitive, and convenient to use for the routine determination of active human renin during its isolation and purification from tissue extracts or from genetically engineered bacterial and nonbacterial expression systems.     

Human renal renin. Complete purification and characterization

Complete purification of human renin from noncancerous, autopsied kidneys is reported. A 480,000-fold purification was achieved to yield renin with a specific activity of 950 Goldblatt units/mg. This preparation satisfied multiple criteria of purity as tested by polyacrylamide gel electrophoresis, isoelectric focusing, specific activity, analytical ultracentrifugation, and immunodouble diffusion. The molecular weight of the pure enzyme determined by sedimentation equilibrium is 40,000. The apparent molecular weight estimated by gel filtration is 41,000. The enzyme has an isoelectric point of pH 5.7. Human renin shows an affinity for concanavalin A, suggesting the presence of carbohydrates. These properties and the amino acid composition of human renin are different from those of renin obtained from other mammalian species. Human renin antibodies prepared with the pure enzyme preparation showed negligible cross-reactivity with renin from other mammalian species. The activity with homologous human renin substrate has a pH optimum of 6, whereas with substrates from other mammalian species the optima were in higher or lower pH ranges.     

Renin substrate in granules from rat kidney cortex.

1. Subcellular fractions of rat kidney cortex generated angiotensin I continuously over 2h when incubated at 37degreesC with rat renin, indicating the presence of renin substrate within cells in the renal cortex. 2. Renin substrate was located in highest specific concentration in particulate fractions. The particles containing renin substrate had a sedimentation velocity slightly lower than mitochondria and renin granules but greater than the microsomal fraction. 3. Isopycnic gradient centrifugation indicated a density of 1.190g/ml for the particles containing renin substrate, compared with 1.201 for renin granules, 1.177 for mitochondria, and 1.170 and 1.230 for lysosomes in the heavy-granule fraction. 4. In the liver, renin substrate was also found in particles, but these had a lower sedimentation rate than those from the kidney. 5. The molecular weights of renin substrate in kidney and liver granules and rat plasma were similar, namely 61000-62000. 6. On the basis of these biochemical findings, a mechanism for the intrarenal production of angiotensin, incorporating a subcellular reaction scheme, is proposed.     

Formation of angiotensin II by tonin from partially purified human angiotensinogen

The renin substrate (angiotensinogen) has been purified from outdated human blood bank plasma. A 100-fold purification was achieved by ammonium sulphate protein fractionation and four successive chromatographic procedures. We show that tonin, a serine protease enzyme found in submaxillary glands of the rat, cleaves the human plasma angiotensinogen, devoid of tonin inhibiting factor(s), at a pH optimum of 5--5.5. It generates a pressor substance that was identified as angiotensin (A) II. The rate of cleavage of the human angiotensinogen preparation by 1 nmol of renin or tonin was calculated to be 1320 nmol AI/h for renin and 26 nmol AII/h for tonin.