Adrenal and vascular renin-like activity in chronic 'two-kidney, one-clip' hypertensive rats

In order to study the vascular and adrenal renin angiotensin system in the chronic phase (4 months after clipping) of 'two-kidney, one-clip' hypertension in rats, systolic blood pressure, plasma renin activity, and tissue renin-like activity in both aorta and adrenal have been measured. Renin activity in adrenal gland was studied in both the zona glomerulosa (GLO) and the remainder of the gland. Results showed an increase in vascular renin activity in chronic hypertensive rats. Moreover it was found that GLO of hypertensive rats presented a significant increase in renin-like activity compared with controls (349.43 +/- 43.86 versus 167 +/- 34.25 ng AI/g/20 h, p less than 0.01) and the fasciculata-reticular-medullar (FRM) portion also showed greater renin activity (345.16 +/- 64.36 versus 57.90 +/- 4.83 ng AI/g/20 h, p less than 0.01). The higher levels of vascular and FRM renin-like activity in chronic renal hypertension are probably a consequence of plasma renin increase. This hypothesis is supported by the fact that bilateral nephrectomy in normal rats induces a significant decrease in plasma renin activity and both aortic and FRM renin-like activity. On the other hand the GLO renin-like activity could depend on both plasma renin and local synthesis since bilateral nephrectomy induces an increase in the renin-like activity in this tissue. These data support the idea that aortic and FRM renin are, at least in part, due to plasma renin uptake and GLO renin is an autonomic system.     

A coupled morphological and biochemical study on the cellular localisation of the intra-adrenal renin granules in rats.

The effects of prolonged (3-week) sodium restriction on the rat zona glomerulosa (ZG) were investigated by ultrastructural stereological and biochemical techniques. The plasma level of aldosterone was increased, and this was coupled with a notable decrease in the volume density (Vv, microns 3/100 microns 3 of cell) of lipid droplets in ZG cells. Renin-like activity (RLA) underwent a significant rise in ZG, and Vv of dense bodies significantly rose in ZG cells. Since RLA and dense-body Vv displayed a highly significant linear correlation (r = 0.884; n = 22, P less than 0.01), the hypothesis is advanced that part of the dense bodies may be granules of prorenin or renin, and that ZG parenchymal cells are directly involved in the intra-adrenal renin production.     

Glucose,insulin and renin activity after sodium loading and depletion in Vipera aspis

Sodium, potassium, chloride, glucose, insulin and renin activity were investigated in fasted Vipera aspis subjected for 3 days to administration of 3% NaCl 5 ml, or injection of a diuretic and water loading to produce sodium depletion. After sodium loading, plasma sodium and glucose were significantly elevated if compared with those of controls, while plasma renin-like activity and plasma insulin were depressed. The insulin and somatostatin producing cells (B- and D-cells) showed only a weak immunoreactivity, while in the glucagon producing cells (A-cells) the immunoreactivity was stronger if compared with the handled controls. After sodium depletion, plasma sodium and glucose were significantly depressed and plasma renin-like activity and plasma insulin were significantly elevated. A strong immunoreactivity was present in B- and D-cells and only a weak immunoreactivity was detectable in the A-cells. These data suggest that the secretory activity of the endocrine pancreas and kidney may be affected, in vipers, by sodium and/or volume status.     

Multiple forms of immunoreactive renin in human pituitary tissue

Immunoreactive renin was demonstrated in pituitary tissues of postmortem human subjects with different diseases. The specific immunoreactive renin activity comprised the majority of the tissue renin-like activity (mean, 83%), indicating the absence of nonspecific actions of proteases such as cathepsin D. We used three pituitary specimens with high levels of the specific renin activity for further biochemical characterization of the enzyme. Small differences were found in the molecular mass (45 K, 42 K and 37 K), binding to concanavalin A-Sepharose, and isoelectric points (pI) (4.72, 4.78, 4.86, 5.06, 5.28 and 5.44). These results seem to be interpreted as evidence for the presence of specific renin in the human pituitary with microheterogeneity.     

Vascular renin-like activity in aorta and mesenteric artery of the rat

Vascular renin-like enzymatic activity (VRLA) has been measured in the artery wall of control and experimental rats. The following groups have been studied: 1-normal salt diet; 2-high salt diet; 3-low salt diet; 4-bilateral nephrectomy (Nx); 5-sham operated for Nx. VRLA was evaluated in the aorta (ARLA) and in the mesenteric arteries (MRLA). Blood samples were obtained for plasma renin activity (PRA) determination. High salt diet decreased PRA, ARLA and MRLA whereas low salt diet increased PRA, did not change ARLA and decreased MRLA. PRA was almost undetectable in Nx animals while ARLA showed a 40% reduction and MRLA was unchanged in these animals. These results would indicate that the changes in PRA induced different variations in the renin-like content of the aorta and the mesenteric artery. The differences could be mainly due to two factors: 1) the capacity of the vascular tissue to bind circulating renin and 2) the capacity of each tissue to synthetize renin-like material in situ.     

Characterization of renin-like activity in human plasma protein IV-4 fraction.

Cohn's fraction IV-4 of human plasma protein generates by itself a substance with slight pressor activity when incubted without any additives. This renin-like activity was readily inactivated by alkaline treatment. Furthermore, the generated pressor substance, when purified, was found very similar to [Aspl]-[Ile5]-angiotensin-I in the following characteristics; 1) heat-stability, 2) dializability, 3) inactivation by trypsin, 4) Rf value and 5) electrophoretic mobility at various pHs, 6) marked enhancement in pressor and oxytocic activities after incubation with normal human plasma or rabbit's lung extract, and 7) cross reactivity with [AspI]-[Ile5]-angiotensin-I in radioimmunoassay. It is concluded that renin-like activity in the preparation of fraction IV-4 of human plasma protein must be renin or an extremely similar enzyme.     

Isolation of a renin-like enzyme from the leech Theromyzon tessulatum

This article reports the purification of a renin-like enzyme (an aspartyl protease) from head parts of the leech Theromyzon tessulatum. After four steps of purification including gel permeation and anion exchange chromatographies followed by reversed-phase HPLC, this enzyme was purified to homogeneity. The renin-like enzyme (of 32 kDa) hydrolyses at neutral pH and at 37°C, the Leu¹⁰-Leu¹¹ bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu¹¹-Val¹²-Tyr¹³-Ser¹⁴ peptide as products, with a specific activity of 1.35 pmol AI/min/mg (K_m 22 μM; K_cat 2.7). The hydrolysis of angiotensinogen is inhibitable at 90% by pepstatin A (IC₅₀ = 4.6 μM), consistent with a renin activity. This is the first biochemical evidence of renin-like enzyme in invertebrates.     

The Renin-Angiotensin System in Fishes

It has been suggested that the renin-angiotensin system (RAS)in mammals may participate in the control of blood pressure,regulation of aldosterone secretion, or in renal functions byinfluencing intrarenal hemodynamics, or possibly by directlyaltering renal tubular sodium reabsorption. Comparative studieshave shown that this system is present among most vertebrates.Renal renin activity and juxtaglomerular cells (JGC), the possiblesite of formation and accumulation of renin, have not been foundin the cyclostomes and elasmobranchs. They seem to have evolvedin primitive bony fishes, being present in all living groupsof actinopterygians and sarcopterygians. Both renin and JGCmay also exist in a holocephalian, the ratfish, Hydrolagus colliei.The functions of the RAS are not yet denned in fishes. Thereis no clear evidence for sodium retaining function of the RASin fishes. Fish angiotensins (angiotensin-like substances) havechemical properties that differ from those of mammals and othertetrapods. It is possible that they also serve quite differentfunctions in fishes than in mammals.     

Cerebral renin-like activity in different functional conditions (author's transl)]

Using the Boucher micromethod of determining renin activity in the frontoparietal cortex, brain stem, hypothalamus, hypophysis, and epiphysis rat tissue in different functional conditions, we have observed the following: 1 In rats previously treated with an I. V., injection of 1-1.5 ml 10% NaCl, an increase of renin-like activity was found in the cerebral cortex, brain stem and hypothalamus, whereas in the grandular tissue there was a significant decrease...     

Effects of angiotensin II and angiotensin II antagonist saralasin on cell growth and renin in 3T3 and SV3T3 cells.

Components of the renin-angiotensin system were studied in established cell culture lines of 3T3 and SV3T3 mouse fibroblasts. The renin content in 3T3 cells was significantly higher than in virus-transformed SV3T3 cells. With time after infection, renin decreased in Simian virus 40 transformed cells, while it increased steadily in mock-infected 3T3 cells. In contrast to renin, angiotensinase activity was higher in SV3T3 cells. Angiotensin II stimulated cell proliferation in 3T3 mouse fibroblasts and decreased their renin content in a dose-related manner. In contrast, saralasin, an angiotensin receptor antagonist, inhibited cell growth in 3T3 and SV3T3 cells and caused an increase of cellular renin concentration. The angiotensin fragments angiotensin (2-8) heptapeptide and angiotensin (4-8) pentapeptide had no effect on cell growth. A significant negative correlation was found between cell proliferation and renin levels in 3T3 and SV3T3 cells irrespective of the treatment. Our results indicate (1) that angiotensin II may be involved in cell growth regulation, (2) that a negative feedback exist between angiotensin II added and intracellular renin content, and (3) that virus infection causes a decrease in intracellular renin synthesis, while non-specific angiotensinase activity is increased under this condition.     

Nature of the pressor substance in rabbit placenta

1. The renin-like substance isolated from the placenta of the rabbit produces a prolonged increase of blood pressure in the nephrectomized rat. If incubated with renin substrate from ox serum, it forms a pressor substance that elevates blood pressure in exactly the same way as does angiotensin. 2. This angiotensin-like principle was concentrated by means of ion-exchange chromatography and compared with Val-5-angiotensins I and II in two paper-chromatography systems and in paper electrophoresis. 3. In all the three methods the unknown principle behaved like a mixture of the two reference compounds. 4. It is concluded that incubation of the renin-like substance of placental origin with substrate from ox serum gave a mixture of Val-5-angiotensins I and II. This is evidence that the enzyme isolated from the placenta is either closely related to, or identical with, renin.     

M protein of a Streptococcus dysgalactiae human wound isolate shows multiple binding to different plasma proteins and shares epitopes with keratin and human cartilage

Besides group A (GAS), Lancefield group C beta-haemolytic streptococci (GCS) have been implicated as a causative agent in outbreaks of purulent pharyngitis. In this study we have investigated a class CI M protein of a Streptococcus dysgalactiae1:256, revealed that 26% of these sera showed serological cross-reactivity between a 68-kDa cartilage protein and the N-terminal part of MC. Only 8% of the sera of healthy patients showed this property. In additional, MC also cross-reacted with antibodies recognising epidermal keratins. The cross-reacting 68-kDa protein from cartilage was different from human serum albumin, but was recognised with anti-vimentin immune serum. The MC was cloned and the gene sequenced. By using PCR, recombinant gene fragments encoding characteristic peptide fragments of MC were expressed in Escherichia coli. The peptides were used to map the binding sites for plasma proteins and to locate the cross-reacting epitopes on the MC molecule. In consequence, sequence alignments revealed that MC shared homologous regions with vimentin and different keratins. Our data, obtained with MC, suggest that not only infections with GAS but also infections with GCS and possibly GGS (the latter species can also produce class CI M-like proteins) may be responsible for the formation of streptococcal-associated sequel diseases.     

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.     

New developments in our knowledge of the chemistry of renin.

Although the role of renin in hypertension continues to be incompletely defined, recent progress in the chemistry of renin has been considerable. Extensive purifications of hog kidney renin and the renin-like mouse submaxillary gland enzyme have been achieved. Various inhibitory peptides based on tetradecapeptide renin substrate have been useful in renin kinetic studies and in renin affinity chromatography. Classification of renin as an acid protease results from its marked inhibition by pepstatin and from the discovery that free carboxyl at the active site is essential for activity in human and hog kidney and mouse submaxillary gland enzymes. The presence of pseudorenin in all tissues has limited the use of model peptides as renin substrates in plasma and crude tissue extracts, since the proteolytic properties of the two enzymes are nearly identical. The existence of renin in multiple, chromatographically separable forms has been known. More recently inactive forms have been found in plasma, amniotic fluid, and hog and rabbit kidneys. Prolonged storage or treatment with acid, trypsin, or pepsin causes activation; in some instances the conversion is from a higher than normal molecular weight. The implications of these findings with respect to the renin-angiotensin system need much further investigation.     

Comparative physiology of the renin-angiotensin system.

Renin or a renin-like substance is found in the kidneys of many vertebrate species. It is absent from the kidneys of cyclostomes and elasmobranchs and first appears in holosteans and the bony fishes as well as in all higher vertebrate species. Juxtaglomerular cell granules also appear first in holosteans and the bony fishes while the macula densa first appears in amphibians. In telecost fishes, the corpuscles of Stannius contain Bowie-stainable granules and a renin-like pressor substance. Among classes and, in some cases, species of vertebrates, specificity in the reaction of renin with a substrate has been demonstrated. There is also some species and class variation in the angiotensin molecule since angiotensins of fishes, amphibians, reptiles and birds have chemical characteristics different from each other and from those of ammmals. A role for renin in stimulating interrenal gland steroid biosynthesis and in influencing water and ion regulation in nonmammalian vertebrates is discussed.     

Multiple forms of human renin. Purification and characterization.

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.