Immunoreactive Angiotensin II Research Articles

Urinary angiotensinogen reflects the activity of intrarenal renin-angiotensin system in patients with IgA nephropathy

A potential contribution of local activation of the renin-angiotensin system (RAS) to the pathogenesis of renal injury has been indicated by evidence for blood pressure-independent renoprotective effects of angiotensin II (AngII) receptor blockers (ARBs). The present study was performed to test the hypothesis that urinary angiotensinogen provides a specific index of intrarenal RAS status in patients with immunoglobulin A (IgA) nephropathy. This paper is a survey of urine specimens from three groups: healthy volunteers, patients with IgA nephropathy and patients with minor glomerular abnormality (MGA). Patients with hypertension, diabetes, reduced glomerular filtration rate and/or who were under any medication were excluded from this study. Urinary angiotensinogen levels were measured by a sandwich enzyme-linked immunosorbent assay system. Urinary angiotensinogen levels were not different between healthy volunteers and patients with MGA. However, urinary angiotensinogen levels, renal tissue angiotensinogen expression and AngII immunoreactivity were significantly higher in patients with IgA nephropathy than in patients with MGA. Baseline urinary angiotensinogen levels were positively correlated with renal angiotensinogen gene expression and AngII immunoreactivity but not with plasma renin activity or the urinary protein excretion rate. In patients with IgA nephropathy, treatment with an ARB, valsartan (40 mg/day), significantly increased renal plasma flow and decreased filtration fraction, which were associated with reductions in urinary angiotensinogen levels. These data indicate that urinary angiotensinogen is a powerful tool for determining intrarenal RAS status and associated renal derangement in patients with IgA nephropathy.

Immunohistochemical distribution of regulatory peptides in the human fetal adenohypophysis

We have studied here the cellular distribution of several regulatory peptides in hormone-producing cells of the human pituitary during the fetal period. Immunohistochemistry was used to show the expression of several regulatory peptides, namely Angiotensin-II, Neurotensin and Galanin, at successive gestational stages and their co-localization with hormones in the human fetal adenohypophysis. Somatotrophs, gonadotrophs and thyrotrophs were differentiated earliest. At gestational week 9, Angiotensin-II immunoreactivity was co-localized only with growth hormone immunoreactivity in somatotrophs, one of the first hormone-producing cells to differentiate. This co-localization remained until week 37. Neurotensin immunoreactivity was present in gonadotrophs and thyrotrophs in week 23, after FSH and TSH hormone differentiation. Galanin immunoreactivity was present in all hormone-producing cell types except corticotrophs. The different pro-opiomelanocortin-derived peptides were detected at different stages of gestation and adrenocorticotrophic hormone immunoreaction was the last to be detected. Our results show an interesting relationship between regulatory peptides and hormones during human fetal development, which could imply that these peptides play a regulatory role in the development of pituitary function.

Angiotensin II and the transcription factor Rel/NF-κB link environmental water shortage with memory improvement

One of the essential requirements even in the most ancient life forms is to be able to preserve body fluid medium. In line with such requirement, animals need to perform different behaviors to cope with water shortages. As angiotensin II (ANGII) is involved on a widespread range of functions in vertebrates, including memory modulation, an integrative role, in response to an environmental water shortage, has been envisioned. Previous work on the semi-terrestrial and brackish-water crab Chasmagnathus granulatus showed that endogenous ANGII enhanced an associative long-term memory and, in addition, that high salinity environment induces both an increase of brain ANGII levels and memory improvement. Here, we show that in the crab Chasmagnathus air exposure transiently increases blood sodium concentration, significantly increases brain ANGII immunoreactivity, and has a facilitatory effect on memory that is abolished by a non-selective ANGII receptor antagonist, saralasin. Furthermore, Rel/NF-κB, a transcription factor activated by ANGII in mammals and during memory consolidation in Chasmagnathus brain, is induced in the crab’s brain by air exposure. Moreover, nuclear brain NF-κB is activated by ANGII, and this effect is reversed by saralasin. Our results constitute the first demonstration in an invertebrate that cognitive functions are modulated by an environmental stimulus through a neuropeptide and give evolutionary support to the role of angiotensins in memory processes. Moreover, these results suggest that angiotensinergic system is preserved across evolution not only in its structure and molecular mechanisms, but also in its capability of coordinating specific adaptative responses.

Neurotransmitters and neuropeptides in the baroreceptor reflex arc: connections between the nucleus of the solitary tract and the ventrolateral medulla oblongata in the rat.

The primary baroreceptor area (nucleus of the solitary tract-NTS) is anatomically interconnected with the rostral ("vasomotor area") and the caudal ("vasodepressor area") ventrolateral medulla by a well-defined arc of neuronal pathways. The chemical character and the direction of these pathways have been investigated with immunohistochemical and neurochemical techniques in intact and brainstem-operated rats. The transection of the neuronal arc resulted in an accumulation of tyrosine hydroxylase immunoreactivity in a small group of cells in the NTS adjacent to the area postrema, ipsilateral to the knife cut. Decreased angiotensinogen mRNA and atrial natriuretic peptide concentrations were measured in the ventrolateral medulla after the cut, and an accumulation of angiotensin II-immunoreactivity was found in neuronal perikarya in the ipsilateral NTS. Intracranial vagotomy caused marked depletions in glutamate levels in the subcommissural portion of the NTS and in the caudal ventrolateral medulla but nowhere else in the brainstem investigated including the rostral ventrolateral medulla.

Measurement of Individual Plasma Angiotensins in Normal Pregnancy and Pregnancy-Induced Hypertension*

Individual angiotensin peptides were measured by a high pressure liquid chromatography-RIA (HPLC-RIA) technique in the plasma of 20 nonpregnant women, 17 women with normal pregnancy, and 49 with pregnancy-induced hypertension. Immunoreactive angiotensin-II (ANG-II) consisted mainly of ANG-(1-8) octapeptide (greater than 65%), variable proportions (15-25%) of ANG-(4-8) pentapeptide, and small to negligible proportions of ANG-(2-8) heptapeptide and ANG-(3-8) hexapeptide. Levels of ANG-(1-8) were significantly higher in women with normal pregnancy than in both nonpregnant women (P less than 0.0006) and women with pregnancy-induced hypertension (P less than 0.008); in the latter, levels were lower with increasing severity of disease. Levels of ANG-(4-8) were higher in women with normal pregnancy than in women with pregnancy-induced hypertension or nonpregnant women. When expressed as a proportion of ANG-(1-8) levels, however, ANG-(4-8) levels were not higher in normal pregnancy than in women with pregnancy-induced hypertension or nonpregnant women. It is concluded that the well known increase in ANG-II levels in normal pregnancy relates predominantly to the active ANG-(1-8) octapeptide and to a far lesser extent to the smaller ANG peptides. Similarly, lower ANG-II levels in pregnancy-induced hypertension relate predominantly to lower ANG-(1-8) levels.

Changes in central and peripheral renin-angiotensin system after furosemide injection.

To examine the effects of acute stimulation on the peripheral and central renin-angiotensin system, simultaneous sampling of blood and cerebrospinal fluid (CSF) for measurements of plasma renin activity (PRA), plasma angiotensin I-immunoreactivity (PAng I-ir), plasma angiotensin II-immunoreactivity (PAng II-ir), plasma angiotensinogen and cerebrospinal fluid angiotensin II-ir (CSF Ang II-ir) and CSF angiotensinogen was carried out following intravenous injection of furosemide (5 mg/kg) in conscious dogs. Administration of furosemide induced marked increases in PRA, Ang I-ir, PAng II-ir and CSF Ang II-ir, however, neither plasma nor CSF angiotensinogen was changed. Furthermore, a relatively large dose (20 mg/kg/min) of intravenously infused synthetic Ang II for 20 min produced a five-fold increase in PAng II-ir compared with no significant increase in CSF Ang II-ir. In spite of significant suppression of PRA and PAng I-ir, there were no significant changes in either plasma or CSF angiotensinogen. These results primarily suggest that the peripheral and the brain renin-angiotensin systems may be linked and that acute changes in the peripheral renin-angiotensin system do not alter either plasma or CSF angiotensinogen.

Catecholamines, Angiotensin II and Sodium Concentrations in Cerebrospinal Fluid in Young Men with Borderline Hypertension

To evaluate the role of central nervous mechanisms and their relationships to the peripheral sympathetic nervous system in borderline hypertension, we measured catecholamines, angiotensin II (AII) and sodium (Na) concentrations in cerebrospinal fluid (CSF) with plasma catecholamines concomitantly in 12 young men with borderline hypertension and 7 age-matched healthy normotensive men on ordinary salt intake. Plasma norepinephrine (NE) and epinephrine (E) were higher in the borderline hypertensives than in the normotensives (NE: 239 +/- 15 vs 190 +/- 11 pg/ml, p less than 0.05, E: 83 +/- 9 vs 43 +/- 6 pg/ml, p less than 0.01). NE levels in CSF were also higher in the borderline hypertensives than in the normotensives (200 +/- 15 vs 150 +/- 18 pg/ml, p less than 0.05). In most of the subjects, CSF E and plasma and CSF dopamine levels were below the sensitivity of the assay. CSF NE correlated positively with both plasma NE (p less than 0.01) and mean blood pressure (p less than 0.05) in all subjects. Immunoreactive AII and Na concentrations in CSF did not differ between the borderline hypertensives and normotensives. These results suggest that peripheral sympathoadrenal overactivity in young subjects with borderline hypertension may be related to an altered function of central noradrenergic neurons. AII and Na in the central nervous system do not appear to have an important role in borderline hypertension.

Alterations in plasma and cerebrospinal fluid norepinephrine and angiotensin II during the development of renal hypertension in conscious dogs.

The levels of norepinephrine (NE), epinephrine (E), and angiotensin II immunoreactivity (AIIir) in plasma and in cerebrospinal fluid (CSF) were measured in eight conscious dogs before and during a 28-day period in the development of two-kidney, one clip (2K1C) hypertension produced by a two-step procedure. The early phase (less than 7 days) of hypertension following partial constriction of the renal artery was accompanied by tachycardia and increases in concentrations of NE and AIIir in both plasma and CSF; E did not change. One week later blood pressure remained elevated (107 +/- 2 after vs 88 +/- 2 mm Hg before clipping, p less than 0.05), but other variables returned to control values. Occlusion of the partially constricted renal artery caused severe hypertension that was initially associated with a transient decrease in levels of NE in both plasma and CSF and a sustained rise in plasma and CSF concentrations of AIIir that persisted for as long as 2 weeks after the second operation. None of these effects was seen in nine sham-operated dogs. Since activation of the renal pressor system is associated with time-related changes in the concentrations of NE and AII in both plasma and CSF, these observations indicate early involvement of both sympathetic and renin-angiotensin systems in the pathogenesis of renovascular hypertension.

Angiotensin II immunoreactivity coexists with renin in the juxtaglomerular granular cells of the kidney.

The multiple physiologic functions of angiotensin II(AII) are generally supposed to be mediated by the peptide generated in the blood circulation. In addition to this extracellular mechanism of AII formation, we have obtained immunohistochemical evidence for the intracellular synthesis of AII in the kidney. Rats were perfused with fixative, and paraffin sections of the kidneys were processed with antisera against renin (EC 3.4.99.19), AII, and other components of the renin--angiotensin system. Renin immunoreactivity was regularly observed in the epithelioid granular cells in the media of the afferent vessel of the glomerulus. AII immunoreactivity was found to coexist within the same cells. This observation points to an intracellular production of AII in the juxtaglomerular epitheloid granular cells. AII may then be released concomitantly with renin in the interstitial fluid and in the blood. The paracrine secretion of AII could exert a local regulatory influence on the tonus of the glomerular vessels.

Effects of water deprivation on immunoreactive angiotensin II levels in plasma, cerebroventricular perfusate and hypothalamus of the rat

To examine whether endogenous angiotensin--which has been suggested to produce increased vasopressin (ADH) release and water intake under dehydration, by stimulating the central nervous system--is derived from the brain or from the circulating blood or from both, the effects of water deprivation for 46 h on immunoreactive angiotensin II (AII) concentrations of plasma, cerebroventricular perfusate and the hypothalamus were studied in conscious and urethane-anaesthetized rats. Immunoreactive AII in plasma and the hypothalamus was extracted with acetone and petroleum ether preceding the determination by radioimmunoassay. The water deprivation significantly increased plasma immunoreactive AII concentration (P less than 0.002) together with plasma osmolality and sodium concentration, and reduced the potassium concentration. However, neither the immunoreactive AII concentration of the ventricular perfusate nor that of the hypothalamus was affected. Both the perfusate and the hypothalamus were very poor in immunoreactive AII (less than 35.0 pg/ml and less than 46.7 pg/g wet tissue, respectively). These results may suggest that increased ADH release and water intake under dehydration are brought about by the angiotensin formed in the circulating blood rather than in the brain.

Presence of angiotensin II in neurons cultured from fetal rat brain

By means of a specific antiserum to angiotensin II (ANGII), it has been possible to demonstrate the existence of ANGII immunoreactivity in cells cultured from fetal rat brain. Dissociated cells cultured from the whole brains of 20-day-old fetuses were stained according to the peroxidase antiperoxidase method. Immunoreactive ANGII was observed in less than 1% of the total cell population. These cells have been identified as neurons on the basis of morphological criteria. Positively stained neuronal processes with localized concentrations of ANGII immunoreactivity were visualized in association with ANGII-containing soma. The presence of ANGII in cultured brain cells provides an experimental model for investigating the role of angiotensin in the CNS.

Angiotensin II immunohistochemistry of the rat brain.

Immunoreactive angiotensin II (AII) in rat brain is identified histochemically by the unlabelled antibody-enzyme method. Addition of protease inhibitors, phenylmethyl sulfonyl fluoride (PMSF) and ethylenediamine tetra-acetic acid (EDTA), to subzero (-10 degrees C) fixation with propylene glycol and formaldehyde provides reproducible preservation of immunoreactive AII. Synaptic boutons within the brainstem and cerebellum contain immunoreactive AII. The deep cerebellar nuclei have the richest density of AII positive synapses. Other nonneuronal cells such as pinealocytes, pituicytes, and 3rd ventricular tanycytes contain immunoreactive AII. This dual AII localization suggests that both neurons and blood vessels may be involved in the transport of AII to sensitive sites within the rat brain.

Impermeability of the blood-cerebrospinal fluid barrier for angiotensin II in rats.

1. Anaesthetized, nephrectomized rats were infused intravenously with unlabelled angiotensin II (AII) or with [3H]angiotensin II (3H-labelled AII). The brain ventricular system was perfused with artificial cerebrospinal fluid. The perfusate was collected from the cisterna magna and analysed for AII by radioimmunological and biochemical methods. 2. No increase of immunoreactive AII in cerebrospinal fluid could be shown during intravenous infusion of AII. 3. During intravenous infusions of 3H-labelled AII at pressor doses small amounts of radioactivity were found in cerebrospinal fluid perfusate. 4. The radioactivity of cerebrospinal fluid outflow could not be related to AII.