Conversion between renin and high-molecular-weight renin in the dog.

Two forms of renin, one of mol.wt. 43,000 and the other 60,000, were found in the dog kidney. Conversion between the two forms of renin was reversible at neutral pH. Though the molecular weight of renin in kidney-cortex homogenate was 43,000, it was completely converted into high-molecular-weight renin in the presence of substances that react with thiol groups. On the contrary, stored renin in the granules was the form of normal size (mol. wt. 43,000) regardless of the absence or presence of such substances. The present experiments indicated that renin is stored in the granules as the form of normal size and might be converted into high-molecular-weight renin when it is released from the granules and attached to some substance in the soluble fraction of renal-cortical tissue.     

Purification of human renin substrate.

Renin substrate, angiotensinogen, has been purified from human plasma by methods which permit the processing of large amounts of outdated bank blood. The purified protein is homogeneous by disc gel electrophoresis at pH 9.5. The specific activity of 18 nmol/mg corresponds to a molecular weight of 56,000, while a higher value, 90,000, is found by gel filtration. Chromatography of partially purified renin substrate on DEAE-cellulose in a descending pH gradient shows evidence for the existence of multiple forms. However, some of these forms appear to be lost after chromatography on hydroxylapatite.     

Pure human inactive renin. Evidence that native inactive renin is prorenin

To clarify contradicting observations on the identity of inactive renin and prorenin, inactive renin was completely purified from native human chorion laeve and the culture medium of human chorion cells. A 720,000-fold purification with 14% recovery was achieved from chorion laeve in 6 steps, including immunoaffinity chromatography on a monoclonal antibody to human renin coupled to Protein A-Sepharose CL-4B. A 3,100-fold purification with 40% recovery was achieved from chorion culture medium in 4 steps, including immunoaffinity chromatography. Inactive renin purified from the two different sources migrated as a single protein band with the same molecular weight of 47,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of multiple components that could be resolved by isoelectric focusing. Both had the same pI values which shifted downward upon activation by trypsin; however, relative peak heights were different between the two preparations. The purified inactive renin from chorion laeve was completely inactive and did not bind to pepstatin-aminohexyl-Sepharose; however, that from chorion culture medium was partially active and completely bound to the pepstatin gel, indicating that each molecule is partially activated. Trypsin-activated inactive renins from both sources were identical with human renal renin in terms of pH optimum and Km. Specific activities of trypsin-activated inactive renin from chorion laeve and chorion culture medium were 529 Goldblatt units/mg of protein and 449 Goldblatt units/mg of protein, respectively. Amino acid sequence analysis of both of the purified inactive renin preparations demonstrated a leucine residue at the amino terminus. The sequence of 11 additional amino acids was identical in both and agreed with that predicted from the base sequence of the renin gene. These findings indicate that preprorenin is converted to prorenin following removal of a 23-amino acid signal peptide and that the native inactive renin, whose amino acid sequence commences with Leu-Pro-Thr..., is prorenin.     

Plasma renin, renin substrate and angiotensin II changes following experimental endotoxinaemia.

The effect of a single dose of endotoxin (B. coli, 1.5 mg/kg intravenously) on plasma renin concentration (PRC), renin substrate (PRSC), and angiotensin II (AT II) was studied in rats over a period of 48 hours. All determinations were performed by specific radioimmunoassay. Six and nine hours following endotoxin administration, renin secretion was decreased, whereas at 48 hours a slight increase in the PRC was found. In contrast, a three-fold elevation of the PRSC occurred during the first 24 hour period, attributable to a stimulation of the hepatic biosynthesis as result of corticosterone oversecretion. According to the observed changes in PRC and PRSC, AT II remains unchanged after six and nine hours, whereas a significant increase was detected after 24 and 48 hours. Based on the actual AT II level, the findings emphasize that in the rat the RAS does participate in the later stages of endotoxin stress only.     

Immunocytochemical localization of renin in mouse kidney.

The distribution of renin in mouse kidney was examined in immunohistochemical studies by using an antiserum against pure mouse submaxillary renin and the peroxidase-antiperoxidase (PAP) technique. At antibody dilutions from 1:10(4) to 1:10(6), renin was found in high concentrations in the epitheloid cells of the vasa afferentia and, in lower concentrations, in the wall of some of the vasa efferentia. Renin was also detected in most of the interlobular arteries. Mesangial cells and Goormaghtigh cells were always free of specific staining. At high antiserum concentrations (i.e., dilutions from 1:10(2) to 1:10(4)) specific reaction product was also observed in the apical part of proximal tubule cells. This staining may represent filtered and pinocytozed renin.     

Synthesis of renin substrate by rat liver.

The present study attempts to determine if the isolated rat liver is capable of synthesizing renin substrate from 14C-labelled amino acids added in the perfusate. The renin substrate is characterized via reaction with renin, forming a substance that is subsequently identified as proangiotensin. Extensive evaluation of the reaction product is carried out by using molecular-sieve chromatography, countercurrent distribution, reactivity with converting enzyme, radioimmunological technique and bioassay. The results demonstrate that isolated rat liver perfused with artificial salt solution is capable of synthesizing a protein that reacts with renin to form a radioactive substance indistinguishable from proangiotensin.     

Aldosterone and renin in essential hypertension.

A review of some recent laboratory findings indicates definite disturbances in aldosterone metabolism and regulation in patients with mild essential hypertension: (a) a significant mean increase in plasma aldosterone concentration in patients with mild and stable essential hypertension, in contrast to the absence of any difference in patients with labile borderline essential hypertension when in a normotensive phase, compared with control subjects; and (b) a significant mean decrease in metabolic clearance rate of aldosterone, associated with a 12% decrease in hepatic blood flow and an increased binding of aldosterone to a transcortin-like plasma globulin. The secretion rate of 18-hydroxy-11-deoxycorticosterone is above the upper range of normal in 60% of patients with mild, uncomplicated essential hypertension. The incidence of low-renin hypertension, when age and race are taken into account, is much lower than previously assumed. Unless measurements are repeated over a long period, one or two low values of plasma renin cannot be considered a permanent marker indicating a special category of patients with essential hypertension. Tonin, a new enzyme discovered by Boucher, which forms angiotensin II directly from a plasma protein, from the tetradecapeptide substrate and from angiotensin I, is present in most tissues, but in highest concentration in the submaxillary gland. This enzyme is under the control of beta-adrenergic receptors.     

New renin inhibitors homologous with pepstatin.

Four homologues of pepstatin, the potent but poorly soluble inhibitor of aspartic proteinases, were synthesized by coupling to the C-terminus of the natural pentapeptide the following amino acid residues: L-arginine methyl ester, L-aspartic acid, L-glutamic acid and the dipeptide L-aspartyl-L-arginine. The peptide-coupling reagent we used, benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate, allowed us to obtain readily pure pepstatin homologues with high yields (60-83%). Pepstatylarginine methyl ester and pepstatylglutamic acid were about one order of magnitude more water-soluble than pepstatin. The four homologues and pepstatin were tested in vitro as inhibitors for highly purified pig and human renins acting on the N-acetyltetradecapeptide substrate. The 50% inhibitory concentrations (IC50) of the homologues were ranged from 0.01 to 1 microM against porcine renin at pH 6.0 (pepstatin IC50 approximately 0.32 microM) and from 5.8 to 41 microM against human renin at pH 6.5 (pepstatin IC 50 approximately 17 microM). By three different graphical methods we showed that pepstatin and the four homologues behaved as competitive inhibitors for porcine renin. The most potent inhibitors were pepstatylaspartic acid and pepstatylglutamic acid, with inhibitory constants respectively 2- and 10-fold smaller than that of pepstatin. By coupling glutamic acid to pepstatin, the ratio solubility/Ki was increased by two orders of magnitude.     

Plasma renin activity in diabetic patients.

Plasma renin activity (PRA) in 40 diabetic patients and 42 healthy controls was investigated using the method of Pickens in modification of Serebrovskaja et al. (1967). PRA was slightly lower in the whole group of diabetes but the difference was not significant. The subgroup of 20 maturity-onset diabetics had significantly lower PRA in comparison with 22 controls of similar age, while PRA in juvenile diabetics did not differ significantly from matched controls. In patients without clinical signs and symptoms of microangiopathy PRA was as high as in the controls. In diabetics with microangiopathy PRA was significantly lower. PRA was also lower in patients with longer duration of the disease. The stimulation of juxtaglomerular apparatus with sodium free diet and diuretic drugs resulted in an increase of PRA both in controls and diabetics. This suggests a functional depression of PRA in diabetic patients. In diabetics with ketoacidosis PRA was higher than in control subjects and decreased after disappearance of ketoacidosis. A high level was recorded in a patient with hyperosmolar coma and a very low level in a patient with polyneuropathy and severe orthostatic hypotension. The possible mechanisms involved in the changes of PRA in diabetic patients are discussed.     

Distribution and disappearance rate of submaxillary renin.

Highly purified submaxillary renin (SR) labeled with 125I was injected intravascularly into adult male mice following removal of submaxillary glands and kidneys, and the disappearance of this labeled SR from the circulating vascular volume was studied on the basis of a two compartment system. There was a fast and a slow component to the disappearance curves. Mean half-times of the fast and slow component were 12.4 +/- 0.4 min and 86 +/- 3 min in sialoadenectomized mice, while in mice whose submaxillary glands and kidneys were removed the half-times were 14.7 +/- 0.4 min and 108 +/- 7 min, respectively. The uptake of radioactivity by various organs of the mouse was also measured. Accumulation of radioactivity occurred in the kidneys and liver. Only trace amounts of radioactivity were found in the other organs. The findings suggest that the fast component of the disappearance curve was probably due to equilibration of the injected labeled SR in the circulation. However, the fast component may be related to some extent to the rapid uptake of labeled SR by the kidneys. The half-time of the slow component may represent the true halflife of SR in mice, since a significant reciprocal relationship between the half-times of the slow component and metabolic rate constant k10 was observed both in sialoadenectomized mice and in nephrectomized-sialoadenectomized mice.     

Prostaglandins and renin release: I. Stimulation of renin release from rabbit renal cortical slices by PGI2.

Prostaglandins have been shown to be involved in the mechanism of renin secretion in a variety of situations. Both arachidonic acid and prostaglandin endoperoxide have been shown to release renin from cortical slices and to be converted to PGI2 by cortical microsomes. In the present studies PGI2 was found to cause a time dependent increase in renin release from rabbit renal cortical slices, a system isolated from any indirect effects that result from the administration of prostaglandins in vivo. The stimulation was linear up to 30 minutes and effective over a range of concentrations from 10(7 M to 10(-5) M. At similar concentrations 6-keto-prostaglandin F1alpha was not active on these slices. Thus, it is proposed that PGI2 exerts a direct effect on the release of renin from cortical cells and may be the mediator of arachidonate or prostaglandin endoperoxide stimulated renin secretion.     

Effects of blood viscosity on renin secretion.

The effects of alterations in blood and plasma viscosities on plasma renin activity (PRA) were studied in dogs anesthetized with pentobarbital. Blood viscosity was altered by changing the hematocrit (Hct) level by isovolemic exchange using packed red blood cells or plasma. Plasma viscosity was elevated by isovolemic exchange using Hct-matched blood with high molecular weight dextran (Dx, mean m.w. approximately 450,000) dissolved in plasma. Following control measurements of plasma and blood viscosities, plasma [Dx], PRA, Hct and hemodynamic functions, the dog was subjected to isovolemic exchange transfusions to either alter the Hct or administer the Dx. Various measurements were repeated 40-60 min after each exchange. Arterial pressure and renal blood flow remained relatively constant after exchanges; increases in plasma and blood viscosities were accompanied by a decrease in renal vascular hindrance (vasodilation) to keep the renal flow resistance at control level. PRA rose with increases in plasma [Dx] and viscosity, and the rise in PRA was best correlated with the decrease in renal hindrance. The changes in PRA and renal hindrance have the same regression line whether blood viscosity was altered by Hct variation or Dx administration. The results indicate that increases in viscosity cause a compensatory vasodilation of renal vessels to cause renin secretion.