Targeting the renin-angiotensin system: what's new?

The renin-angiotensin system is a key target for drugs combating cardiovascular disease. Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor type-1 (AT1 receptor) blockers are well known. However, angiotensin peptides can be generated through a number of pathways besides the classic system. This review outlines some of these pathways, their relation to the classic system and the likely effect of inhibiting them. Renin is still the key enzyme in angiotensin peptide generation and seems to be the only route to angiotensin I formation in vivo. Renin inhibitors may have some advantages in terms of specificity. Also, by blocking angiotensin I generation, the production of downstream bioactive angiotensin I metabolites should also be blocked. Chymase, a mast cell serine protease, cleaves angiotensin I to produce angiotensin II and may be important at sites of inflammation such as atherosclerotic plaque. Angiotensin-converting enzyme 2 (ACE2), a carboxypeptidase structurally related to ACE but resistant to ACE inhibitors, has a protective effect on cardiac function. Neutral endopeptidase 24.11 breaks down both atrial natriuretic peptide and angiotensin II. Inhibiting it potentiates the action of endogenous atrial peptide but only affects circulating angiotensin II when basal levels are above normal. Dual inhibitors of ACE and endopeptidase 24.11 may be of value where there is both sodium retention and increased angiotensin II. Targeting the renin-angiotensin system by gene therapy or antibody treatment may provide a longer-term treatment for hypertension.

The action of salt and captopril on blood pressure in mice with genetic hypertension.

OBJECTIVE: Does captopril lower blood pressure in genetically hypertensive, normotensive and hypotensive mice under normal and salt-loaded conditions? DESIGN AND METHODS: Groups of inbred mice that were genetically hypertensive, normotensive or hypotensive were given one of the following treatments: (a) captopril in drinking water for 7 days; controls were given water. (b) 0.85% saline to drink for up to 14 days; controls were given water. (c) Water or saline followed by captopril/water or captopril/saline for 7 days. (d) In hypotensive mice only, 0.85% saline, 0.85% saline plus captopril, water or captopril in water. Systolic blood pressures (SBP) were measured by a computerized tail-cuff sphygmomanometer. Results were compared by analysis of variance (ANOVA). RESULTS: Captopril lowered SBP in all strains of mice. When saline was given with captopril, the fall in SBP was slower but the final SBP level was similar to that of mice given captopril in water. Hypotensive mice showed a transient rise in SBP on saline, which was abolished by concurrent treatment with captopril. CONCLUSION: Captopril lowers blood pressure in hypertensive, normotensive and hypotensive mice. Salt-loading retards the captopril-induced fall in SBP, but the final level of SBP achieved is similar to that in mice given captopril with water. The BPL1 strain of mouse was slightly salt-sensitive, and this was abolished by captopril.

Polymorphisms of the renin gene promoter in spontaneously hypertensive and Wistar-Kyoto rats.

1. In the present study, 1.39 kb of the renin gene 5' region in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was amplified by polymerase chain reaction from genomic DNA and sequenced. Consistent differences in the renin gene sequence of SHR and WKY rats were found at positions -725, -727, -979 and -1126/-1129 as numbered from the transcription start site (+1). No polymorphism was specific to hypertensive rats. 2. Gel-shift assays were performed using labelled SHR renin promotor DNA and nuclear proteins extracted from rat kidneys. The regions between -1122 and -1139 and between -701 and -797 showed protein binding.

The expression of renin-binding protein and renin in the kidneys of rats with two-kidney one-clip hypertension.

OBJECTIVE: To test the hypothesis that renin-binding protein (RnBP) is involved in modulating the intracellular processing or release of renin, we examined the expression of RnBP in clipped and contralateral kidneys of rats with two-kidney one-clip hypertension, and in left and right kidneys from sham-operated control rats. DESIGN AND METHODS: Kidneys from rats with two-kidney one-clip hypertension and from control rats were either snap-frozen for extraction of mRNA or fixed for in-situ hybridization and immunochemistry. Reverse-transcription polymerase chain reaction on renal mRNA was performed using primers for renin, RnBP, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and angiotensin-converting enzyme (ACE). In addition, renal total RNA was analysed by Northern blotting for RnBP, GAPDH and angiotensin II type 1A (AT1A) receptor mRNA, and the intensity of the bands was measured by laser densitometry. In situ hybridization for renin mRNA was carried out using digoxygenin-labelled antisense oligonucleotides and for RnBP using labelled antisense oligonucleotides and an antisense riboprobe. Controls included sections treated with RNase and sections stained with sense oligonucleotides. RESULTS: The level of expression of mRNA for RnBP is similar in clipped and contralateral kidneys of renal hypertensive rats; in contrast, renin mRNA expression is upregulated in the clipped kidney. Renin-binding protein is expressed mainly in renal tubules and collecting ducts unlike renin, which is expressed in the glomerular afferent arteriole. We did not detect lateralization of expression for ACE or the AT1A receptor between clipped and contralateral kidneys. CONCLUSION: Renin-binding protein expression is unchanged between clipped and contralateral kidneys. Therefore, a physiological stimulus that upregulates renin gene expression in clipped kidneys does not affect RnBP expression. The main sites of RnBP expression are the renal tubules and collecting ducts; in contast renin is expressed at the glomerular pole. The results show that RnBP is not colocalized or coregulated with renin in this model of hypertension.

Reduced vas deferens contraction and male infertility in mice lacking P2X1 receptors.

P2X1 receptors for ATP are ligand-gated cation channels, present on many excitable cells including vas deferens smooth muscle cells. A substantial component of the contractile response of the vas deferens to sympathetic nerve stimulation, which propels sperm into the ejaculate, is mediated through P2X receptors. Here we show that male fertility is reduced by approximately 90% in mice with a targeted deletion of the P2X1 receptor gene. Male mice copulate normally--reduced fertility results from a reduction of sperm in the ejaculate and not from sperm dysfunction. Female mice and heterozygote mice are unaffected. In P2X1-receptor-deficient mice, contraction of the vas deferens to sympathetic nerve stimulation is reduced by up to 60% and responses to P2X receptor agonists are abolished. These results show that P2X1 receptors are essential for normal male reproductive function and suggest that the development of selective P2X1 receptor antagonists may provide an effective non-hormonal male contraceptive pill. Also, agents that potentiate the actions of ATP at P2X1 receptors may be useful in the treatment of male infertility.

Potent in vivo inhibitors of rat renin: analogues of human and rat angiotensinogen sequences containing different classes of pseudodipeptides at the scissile site.

Using solid-phase methodology we have synthesised peptides based on the 8-14 or 6-14 human and rat angiotensinogen sequences, containing the following different isosteric units at the P1-P1' cleavage site: Leu-psi[CH2NH]Leu; Leu-psi[CH(OH)CH2]Val; Leu-psi[CH(OH)CH2]Leu and Leu-psi[CH(NH2)CH2]Val. In vitro, peptide Piv-His-Pro-Phe-His-Leu-psi[CH(OH)CH2]Leu-Tyr-Tyr-Ser-NH2(XXI) is the most potent inhibitor of rat plasma renin reported having an IC50 of 0.21 nM; it is a much weaker inhibitor of human renin (IC50 45 nM). Peptide Boc-His-Pro-Phe-His-Leu-psi[CH(OH)CH2] Leu-Val-Ile-His-NH2 (XX) was a highly effective inhibitor of rat renin in vivo. When infused (1 mg/kg/h) into two-kidney, one-clip chronic renal hypertensive rats, it lowered blood pressure and suppressed both plasma renin and angiotensin II. When given as a bolus (1 mg/kg) there was a divergence between the rapid rebound of renin levels and blood pressure, which remained suppressed. These results indicate that potent in vivo inhibitors of rat renin could be useful not only in examining the role of circulating renin but also in elucidating the equally important involvement of extracirculatory renin pools.

New renin inhibitors containing novel analogues of statine.

Solid-phase methodology has been used to synthesize a series of peptides based on the N-terminal sequence of human angiotensinogen in which statine (Sta) or the novel analogues (3S,4S)-3,4-diamino- or (3R,4S)-3,4-diamino-6-methylheptanoic acid (Ads or R-Ads) and (3S,4S)-4-amino-3-aminomethyl- or (3R,4S)-4-amino-3-aminomethyl-6-methylheptanoic acid (Amd or R-Amd) replace either residue 10 or both residues 10-11 at the P1-P1' cleavage site. The synthesis of these novel analogues of statine together with biological results on the inhibition of human and rat renin by peptides derived from them is reported. The absolute stereochemistry of the (3S,4S) Ads was determined by an X-ray crystallographic analysis of its N gamma-Boc, B beta-Z, R(+)-1-methyl benzamide derivative. Peptide Boc-His-Pro-Phe-His-Sta-Val-Ile-His-NH2 (VI) is the best inhibitor of human renin containing Sta at position 10. However, peptides containing Ads and Amd gave better rat renin inhibitors than the corresponding Sta-containing peptides. Peptides Boc-His-Pro-Phe-His-Ads-Val-Ile-His-NH2 (VII) having Ads at position 10 had an IC50 of 12 nM against rat renin. Although Sta has come to be accepted as an isosteric replacement for a dipeptide unit rather than for a single amino acid residue, in our series of inhibitors Sta is more effective when replacing only the amino acid at position 10 in the natural angiotensinogen sequence. None of the peptides gave any effect in vivo in a hypertensive rat model.

Relation between renin and prorenin in plasma from hypertensive patients and normal people: evidence for different renin:prorenin ratios.

Renin and prorenin concentrations in plasma correlate closely, but the proportion of active renin to prorenin shows some variation in different forms of hypertension. The proportion of active renin to prorenin is higher in normal renin essential hypertensives and in patients with renal hypertension than in normal subjects, and it is lower in low renin essential hypertension and primary hyperaldosteronism. Plasma deficient in plasma prekallikrein shows a lower proportion of active renin than might be expected. Active renin and plasma angiotensin II concentration show a strong correlation while prorenin correlates weakly with plasma angiotensin II concentration.

Renin in Wilms' tumor: prorenin as an indicator.

In a prospective study, 25 children with nephroblastoma (Wilms' tumor) had preoperative plasma prorenin and renin concentrations measured. The mean plasma renin and prorenin concentrations in the patients were raised compared with a control group of patients without nephroblastoma. Four children had recurrence of tumor and, in three, this was associated with an increase in plasma prorenin concentration. Nephroblastoma tissue contained immunoreactive renin and renin messenger RNA, and the renin protein was immunologically and biochemically similar to normal human renin. We conclude that prorenin concentrations in plasma are an indicator of nephroblastoma.

Second messengers involved in the control of renin secretion in cultured human nephroblastoma cells.

We aimed to study the intracellular signaling mechanisms involved in the stimulation and suppression of renin secretion from human nephroblastoma cells. Isoproterenol and forskolin increased intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration and stimulated renin secretion as did the addition of dibutyryl-cAMP. Atrial natriuretic peptide (ANP) suppressed basal renin secretion and increased the concentration of extracellular guanosine 3',5'-cyclic monophosphate (cGMP). When ANP was added in the presence of isoproterenol or forskolin, the increase in cGMP was reduced. ANP attenuated the cAMP response to isoproterenol but not forskolin. Nephroblastoma cell membranes contained the guanosine-binding proteins Gs and Gi2, and the isoforms were similar to those in vascular smooth muscle. A functional role for Gi was indicated because the ANP-induced suppression of basal renin secretion was blocked by pertussis toxin. We conclude that cAMP stimulates and cGMP suppresses basal renin secretion, but neither fully accounts for the suppression of stimulated renin secretion by ANP.

Renal ischaemia, transient glomerular leak and acute renal tubular damage in patients envenomed by Russell's vipers (Daboia russelii siamensis) in Myanmar.

Fifty-two patients who had been bitten by Russell's vipers in Myanmar developed acute renal failure (serum creatinine exceeding 1.3 mg/dL). Thirty-four of them (65%) became oliguric, but the other 18 (35%) maintained a urine output of more than 400 mL/24 h. In oliguric patients, gastrointestinal haemorrhages, renal angle tenderness and conjunctival oedema occurred more commonly, and peak serum creatinine, blood urea nitrogen and the fractional excretion of sodium were significantly higher (P < 0.01) than in non-oliguric patients, indicating a greater degree of renal damage. Urinary concentrations of beta 2 microglobulin and retinol binding protein were raised in most of the patients indicating failure of proximal tubular reabsorption of these proteins, while high urinary N-acetyl glucosaminidase concentrations were consistent with renal tubular damage. Plasma concentrations of active renin were very high, suggesting that renal ischaemia, associated with activation of the renin-angiotensin system, was involved in the development of renal dysfunction.

Hormonal control of atrial natriuretic peptide synthesis and secretion from cultured atrial myocytes.

We have established optimal culture conditions for growing well-differentiated atrial myocytes from newborn rats. These myocytes show the morphological features of myocytes in intact atria; including perinuclear endocrine storage granules, contractile apparatus and numerous mitochondria. The cells synthesise, store and secrete immunoreactive ANP. Stored ANP is of similar molecular weight to pro-ANP (1-126); secreted ANP is the active form ANP (99-126) but processing appears to take place slowly after secretion. ANP secretion is stimulated by endothelin. Synthesis and secretion of ANP are markedly reduced by the beta-adrenergic agonist isoproterenol, and by forskolin.

Dexamethasone-induced differentiation of atrial myocytes in culture.

Atrial and ventricular myocytes from fetal and newborn rats were cultured in medium supplemented with fetal or newborn calf serum with and without glucocorticoid. Myocyte morphology was examined by light and electron microscopy, and the amount of stored and secreted atrial natriuretic peptide (ANP) was measured. Without dexamethasone, neonatal atrial myocytes cultured for 7 days contained myofibrils organized into sarcomeres and numerous endocrine granules containing immunostainable ANP. Secretion of immunoreactive ANP reached a peak between days 7 and 9 of culture. Myocytes from fetal rats secreted ANP but contained few endocrine granules, and myofilaments were poorly organized. By contrast, the addition of dexamethasone (1 nM-1 microM) to the culture medium of newborn myocytes promoted development of numerous endocrine storage granules, mitochondria, and myofibrils with prominent Z-bands. Dexamethasone also increased the cellular content of ANP and ANP-specific mRNA in both atrial and ventricular myocytes. In the presence of dexamethasone myocytes maintained their structural integrity for periods of at least 45 days.

Atrial natriuretic peptide suppresses isoprenaline and dibutyryl cyclic adenosine monophosphate-induced cell growth in cultured renin-secreting human nephroblastoma cells. Comparison with forskolin-induced renin secretion.

OBJECTIVE: Renin secretion in response to long-term exposure to isoprenaline, dibutyryl cyclic adenosine monophosphate (dbcAMP), forskolin and atrial natriuretic peptide (ANP) was measured in cultured human nephroblastoma cells. METHODS: Human nephroblastoma cells in culture were treated long-term (1-12 days) with isoprenaline, dbcAMP or forskolin, alone or in combination with ANP; renin release and cell growth were studied. RESULTS: The increase in renin output caused by isoprenaline and dbcAMP could be accounted for by stimulation of cell growth. The effect of isoprenaline was blocked by propranolol. Forskolin stimulated renin secretion per cell. ANP increased extracellular cyclic guanosine monophosphate and suppressed basal renin output. Suppression of basal renin output was due to a reduced secretion rate per cell, without a change in cell growth. ANP suppressed isoprenaline-induced and dbcAMP-induced renin output by blocking increased cell growth, and forskolin-induced renin output by blocking renin secretion. CONCLUSIONS: These results suggest that beta-receptor agonists and ANP interact within the kidney to control renin secretion, by helping to determine the number of renin-secreting cells.

High blood pressure: hunting the genes.

High blood pressure is a disease of unknown cause. Family history of the disease indicates higher risk, but it is not known which genes are involved or how they interact with environmental influences to produce the disorder. Molecular biology offers an approach to problems that have not so far been solved by classical physiology or biochemistry. By analysing polymorphic variation in chromosome markers such as minisatellite sequences, or by restriction fragment polymorphism analysis of candidate genes, attempts are being made to link genetic variations with hypertension. In genetically hypertensive rats, hypertension is associated with a polymorphism of the renin gene and with other loci on chromosomes 10 and 18. The role of these loci in human hypertension remains to be determined. Other genes such as sodium-lithium countertransport may be involved. Environmental factors such as stress or salt intake could influence the rate or timing of expression of certain genes and thus result in hypertension.

Hypertension associated with increased renin concentrations in nephroblastoma.

An infant with severe hypertension who had a nephroblastoma which was secreting active renin is described. Nephroblastoma must be included in the differential diagnosis of hypertension associated with increased renin concentrations, even in the absence of an abdominal mass.

Renin gene expression in nephroblastoma.

Most cases of nephroblastoma have high plasma levels of prorenin which is biologically inactive. Plasma prorenin levels fall to normal following nephrectomy. In order to ascertain whether renin synthesis occurs in nephroblastomas we decided to search for renin-specific mRNA using a cDNA probe and Northern blot analyses on total RNA purified from snap-frozen human tumour tissue obtained at nephrectomy. We demonstrated renin-specific mRNA in 5/11 (45 per cent) nephroblastomas. It was 1.6 Kb in length, similar to the mRNA detected in normal kidney tissue and in kidneys with renal artery stenosis. In one of the cases of nephroblastoma, in which we could detect no normal renin mRNA at 1.6 Kb, the cDNA probe hybridized with a higher molecular weight mRNA 3 Kb in length. We conclude that some nephroblastomas synthesize renin.

Renin production by nephroblastoma cells in culture.

Nephroblastoma cells have been cultured for 19 days. During this time cell growth and active and inactive renin secretion were monitored. Inactive renin was found to be secreted in excess of active renin. The main secretory protein, precipitated with antirenin IgG was found to have a molecular mass of 52,000 daltons.