Ang-(1-7) treatment attenuates lipopolysaccharide-induced early pulmonary fibrosis.

Early pulmonary fibrosis is the leading cause of poor prognosis in patients with acute respiratory distress syndrome (ARDS). However, whether the renin-angiotensin system (RAS) can serve as a therapeutic target is unknown. In this study, an animal model of early pulmonary fibrosis was established via the LPS three-hit regimen. Afterwards, the animals were treated with intraperitoneal injections of Ang-(1-7), AVE0991, or A779 once per day for 20 days. The plasma and BALF AngII levels of the animals were increased, while there were no significant changes in Ang-(1-7) levels in lung tissue after LPS treatment. Furthermore, the AT1R protein levels were significantly increased and the Mas levels were significantly decreased on days 14 and 21. Administration of Ang-(1-7) downregulated LPS-induced AT1R mRNA expression, which was upregulated by A779. The expression of Mas mRNA responded in the opposite direction relative to AT1R. Moreover, LPS caused decreased levels of Mas and E-cadherin and increased AT1R, Vimentin, and Src phosphorylation levels. Ang-(1-7) or AVE0991 blocked these effects but was counteracted by A779 treatment. Our findings suggested that AngII and AT1R levels exhibit opposite dynamic trends during LPS-induced early pulmonary fibrosis, as do Ang-(1-7) and Mas. Ang-(1-7) exerts protective effects against early pulmonary fibrosis, mainly by regulating the balance between AngII and AT1R and between Ang-(1-7) and Mas and by inhibiting Src kinase activation.

Direct renin inhibition is not enough to prevent reactive oxygen species generation and vascular dysfunction in renovascular hypertension.

Renin-angiotensin system activation promotes oxidative stress and endothelial dysfunction. However, no previous study has examined the effects of the renin inhibitor aliskiren, either alone or combined with angiotensin II type 1 antagonists on alterations induced by two-kidney, one-clip (2K1C) hypertension. We compared the vascular effects of aliskiren (50mg/kg/day), losartan (10mg/kg/day), or both by gavage for 4 weeks in 2K1C and control rats. Treatment with losartan, aliskiren, or both exerted similar antihypertensive effects. Aliskiren lowered plasma Ang I concentrations in sham rats and in hypertensive rats treated with aliskiren or with both drugs. Aliskiren alone or combined with losartan decreased plasma angiotensin II concentrations measured by high performance liquid chromatography, whereas losartan alone had no effects. In contrast, losartan alone or combined with aliskiren abolished hypertension-induced increases in aortic angiotensin II concentrations, whereas aliskiren alone exerted no such effects. While hypertension enhanced aortic oxidative stress assessed by dihydroethidium fluorescence and by lucigenin chemiluminescence, losartan alone or combined with aliskiren, but not aliskiren alone, abolished this alteration. Hypertension impaired aortic relaxation induced by acetylcholine, and losartan alone or combined with aliskiren, but not aliskiren alone, reversed this alteration. Losartan alone or combined with aliskiren, but not aliskiren alone, increased plasma nitrite concentrations in 2K1C rats. These findings show that antihypertensive effects of aliskiren do not prevent hypertension-induced vascular oxidative stress and endothelial dysfunction. These findings contrast those found with losartan and suggest that renin inhibition is not enough to prevent hypertension-induced impaired redox biology and vascular dysfunction.

Mechanism of antihypertensive effect of Mucuna pruriens L. seed extract and its isolated compounds.

Background In the search of safe and effective lead molecules from natural sources, Mucuna pruriens (MP) L. (Fabaceae) seeds were utilized for exploring the antihypertensive potential. Traditionally, it is used as diuretic and hypotensive. Methods Bioassay-guided fractions were utilized for the isolation of active compounds by column chromatography. IC50 value, enzyme kinetics and inhibition mechanism were determined. In vivo time and dose-dependent hypotensive study followed by changes in mean arterial pressure (MAP) induced by angiotensin I (3 nmol/kg), angiotensin II (3 nmol/kg), and bradykinin (10 nmol/kg) in anesthetized rats was done. Plasma and tissue angiotensin I-converting enzyme (ACE) activities were also determined. Results Phytochemical analysis by spectroscopic techniques revealed the presence of known compounds like genistein, ursolic acid and L-DOPA from the ethyl acetate and water fraction, respectively. In vitro study revealed MP ethyl acetate (MPEA) fraction and genistein as the most active fraction (IC50 156.45 µg/mL) and compound (IC50 253.81 µM), respectively. Lineweaver-Burk plots revealed a non-competitive mode of inhibition. ACE protein precipitation was the suggested mechanism for inhibition. The extract showed a time- and dose-dependent decrease in MAP. Genistein was able to dose-dependently reduce the MAP, up to 53±1.5 mmHg (40 mg/kg, i.v.). As compared to control, it showed a dose-dependent decrease in plasma ACE activity of 40.61 % and 54.76 % at 10 mg/kg and 20 mg/kg, respectively. It also decreased the ACE activity in the aorta (107.67nM/ml min at 10 mg, p<0.001; 95.33nM/ml min at 20 mg p<0.001). Captopril was used as a standard for various in vitro and in vivo assays. Conclusions The study revealed the antihypertensive potential of MP seed compounds via ACE inhibition.

Inhibition of soluble epoxide hydrolase does not improve the course of congestive heart failure and the development of renal dysfunction in rats with volume overload induced by aorto-caval fistula.

The detailed mechanisms determining the course of congestive heart failure (CHF) and associated renal dysfunction remain unclear. In a volume overload model of CHF induced by creation of aorto-caval fistula (ACF) in Hannover Sprague-Dawley (HanSD) rats we explored the putative pathogenetic contribution of epoxyeicosatrienoic acids (EETs), active products of CYP-450 dependent epoxygenase pathway of arachidonic acid metabolism, and compared it with the role of the renin-angiotensin system (RAS). Chronic treatment with cis-4-[4-(3-adamantan-1-yl-ureido) cyclohexyloxy]benzoic acid (c-AUCB, 3 mg/l in drinking water), an inhibitor of soluble epoxide hydrolase (sEH) which normally degrades EETs, increased intrarenal and myocardial EETs to levels observed in sham-operated HanSD rats, but did not improve the survival or renal function impairment. In contrast, chronic angiotensin-converting enzyme inhibition (ACEi, trandolapril, 6 mg/l in drinking water) increased renal blood flow, fractional sodium excretion and markedly improved survival, without affecting left ventricular structure and performance. Hence, renal dysfunction rather than cardiac remodeling determines long-term mortality in advanced stage of CHF due to volume overload. Strong protective actions of ACEi were associated with suppression of the vasoconstrictor/sodium retaining axis and activation of vasodilatory/natriuretic axis of the renin-angiotensin system in the circulating blood and kidney tissue.

Hyperbaric oxygenation modulates vascular reactivity to angiotensin-(1-7) in diabetic rats: potential role of epoxyeicosatrienoic acids.

Previously, a facilitating effect of hyperbaric oxygenation (HBO2) on aortic ring responses to angiotensin-(1-7) in healthy rats was reported, with epoxyeicosatrienoic acids (EETs) possibly playing an important role. The aim of this study was to assess whether HBO2 exerts similar effects in diabetic rats and to further explore the role of specific cytochrome P450 (CYP) enzymes in changes induced by HBO2. Aortic relaxation to angiotensin-(1-7) was significantly higher in HBO2 diabetic rats compared to control diabetic rats, while HBO2 had no effect on angiotensin II contraction. N-methylsulphonyl-6-(2-propargyloxyphenyl/hexanamide inhibited the facilitation of angiotensin-(1-7) responses in HBO2 rats, suggesting an important role of EETs in this modulation. mRNA expression of CYP2J3 and protein expression of CYP2C11 were significantly upregulated in HBO2 diabetic rats, whereas CYP4A1, CYP4A2 and CYP4A3 mRNA and CYP2J3 protein expression was similar between groups. Mean arterial pressure, ferric reducing ability of plasma and Thiobarbituric Acid Reactive Substances levels and serum angiotensin-(1-7) concentrations were not significantly changed.

NaCl plus chitosan as a dietary salt to prevent the development of hypertension in spontaneously hypertensive rats.

The effect of NaCl plus 3% chitosan on the systolic blood pressure of spontaneously hypertensive rats (SHR) were evaluated and compared with NaCl plus KCl (NaCl, 49.36% + KCl 49.36%) and chitosan or NaCl treatment alone. In SHR, administration of NaCl plus chitosan (44 mM Na/day) for two months significantly decreased the systolic blood pressure greater than of NaCl plus KCl and NaCl alone. NaCl plus chitosan resulted, though not statistically significant, in decreased urinary Na(+) excretion and decreased blood urea nitrogen levels. Urinary creatinine of NaCl plus chitosan was slightly decreased compared to 3 treated groups. Serum electrolytes levels, however, remained unchanged. The combination of NaCl and chitosan may be superior to the conventional use of NaCl plus KCl or NaCl alone in the prevention of hypertension. Even though these supplementary diets have demonstrated potential anti-hypertensive effects in the experimental animal model, further research is needed before any recommendations can be made.

Angiotensin-(1-7), an alternative metabolite of the renin-angiotensin system, is up-regulated in human liver disease and has antifibrotic activity in the bile-duct-ligated rat.

Ang-(1-7) (angiotensin-1-7), a peptide product of the recently described ACE (angiotensin-converting enzyme) homologue ACE2, opposes the harmful actions of AngII (angiotensin II) in cardiovascular tissues, but its role in liver disease is unknown. The aim of the present study was to assess plasma levels of Ang-(1-7) in human liver disease and determine its effects in experimental liver fibrosis. Angiotensin peptide levels were measured in cirrhotic and non-cirrhotic patients with hepatitis C. The effects of Ang-(1-7) on experimental fibrosis were determined using the rat BDL (bile-duct ligation) model. Liver histology, hydroxyproline quantification and expression of fibrosis-related genes were assessed. Expression of RAS (renin-angiotensin system) components and the effects of Ang-(1-7) were examined in rat HSCs (hepatic stellate cells). In human patients with cirrhosis, both plasma Ang-(1-7) and AngII concentrations were markedly elevated (P<0.001). Non-cirrhotic patients with hepatitis C had elevated Ang-(1-7) levels compared with controls (P<0.05), but AngII concentrations were not increased. In BDL rats, Ang-(1-7) improved fibrosis stage and collagen Picrosirius Red staining, and reduced hydroxyproline content, together with decreased gene expression of collagen 1A1, alpha-SMA (smooth muscle actin), VEGF (vascular endothelial growth factor), CTGF (connective tissue growth factor), ACE and mas [the Ang-(1-7) receptor]. Cultured HSCs expressed AT1Rs (AngII type 1 receptors) and mas receptors and, when treated with Ang-(1-7) or the mas receptor agonist AVE 0991, produced less alpha-SMA and hydroxyproline, an effect reversed by the mas receptor antagonist A779. In conclusion, Ang-(1-7) is up-regulated in human liver disease and has antifibrotic actions in a rat model of cirrhosis. The ACE2/Ang-(1-7)/mas receptor axis represents a potential target for antifibrotic therapy in humans.

Rehmannia glutinosa ameliorates the progressive renal failure induced by 5/6 nephrectomy.

AIM OF THE STUDY: Rehmannia glutinosa, the steamed root of the Scrophulariaceae family, has been widely used in Asian countries for the treatment of renal diseases. In this study, we evaluated the renoprotective effect of aqueous extract of Rehmannia glutinosa in progressive renal failure. MATERIALS AND METHODS: The effects of Rehmannia glutinosa on renal function, 24-h proteinuria, and the expression of angiotensin II, angiotensin II type 1 (AT(1)) receptor, TGF-beta1, and type IV collagen in renal cortex were analyzed in progressive renal failure rats induced by 5/6 nephrectomy. RESULTS: Rehmannia glutinosa reduced the serum creatinine level, 24-h urinary protein excretion, and glomerulosclerosis, and it also inhibited the expression of angiotensin II, AT(1) receptor, TGF-beta1 and type IV collagen in the renal cortex. CONCLUSIONS: These results suggest that the renoprotective effect of Rehmannia glutinosa might be mediated by suppressing the expression of angiotensin II and AT(1) receptor and by regulating TGF-beta1 and type IV collagen expression.

NaCl plus chitosan as a dietary salt to prevent the development of hypertension in spontaneously hypertensive rats

The effect of NaCl plus 3% chitosan on the systolic blood pressure of spontaneously hypertensive rats (SHR) were evaluated and compared with NaCl plus KCl (NaCl, 49.36% + KCl 49.36%) and chitosan or NaCl treatment alone. In SHR, administration of NaCl plus chitosan (44 mM Na/day) for two months significantly decreased the systolic blood pressure greater than of NaCl plus KCl and NaCl alone. NaCl plus chitosan resulted, though not statistically significant, in decreased urinary Na+ excretion and decreased blood urea nitrogen levels. Urinary creatinine of NaCl plus chitosan was slightly decreased compared to 3 treated groups. Serum electrolytes levels, however, remained unchanged. The combination of NaCl and chitosan may be superior to the conventional use of NaCl plus KCl or NaCl alone in the prevention of hypertension. Even though these supplementary diets have demonstrated potential anti-hypertensive effects in the experimental animal model, further research is needed before any recommendations can be made.

Angiotensin II type 1 receptor blockade abolishes specific K(ATP)channel gene expression in rats with myocardial ischemia.

The cardiac ATP-sensitive potassium (K(ATP)) channel is potentially composed of an inward rectifier potassium channel (Kir6.1 and/or Kir6.2) subunit and the cardiac type of sulfonylurea receptor (SUR2A). We reported that cardiac Kir6.1 mRNA and protein are specifically upregulated in the non-ischemic as well as the ischemic regions in rats with myocardial ischemia, suggesting that humoral and/or hemodynamic factors are responsible for this regulation. In the present study, pretreatment with TCV-116, an angiotensin (Ang) II type 1 receptor antagonist, completely inhibited the upregulation of Kir6.1 mRNA and protein expression in both regions of rat hearts subjected to 60 min of coronary artery occlusion followed by 24 h of reperfusion; whereas pretreatment with lisinopril, an Ang converting enzyme (ACE) inhibitor, partly inhibited this upregulation. Except for rats pretreated with TCV-116, Kir6.1 mRNA levels were positively correlated with those for brain natriuretic peptide (BNP), a molecular indicator of regional wall stress, in both the non-ischemic and the ischemic regions. Plasma Ang II levels were not elevated in rats with control myocardial ischemia compared with sham rats. Thus, the stress-related induction of cardiac Kir6.1 mRNA and protein expression under myocardial ischemia is inhibited by pretreatment with an AT1 antagonist, but also in part by an ACE inhibitor, suggesting that activation of local renin-angiotensin system may play a role.

Long-term follow-up of a girl with the neonatal form of Bartter's syndrome.

We followed up a girl with the neonatal form of Bartter's syndrome for sixteen years and determined the sensitivity to angiotensin II before and during the indomethacin treatment. A 4-month-old girl was admitted to our hospital, because of severe hypokalemia and growth retardation. Initially we treated her with spironolactone and potassium supplements. This treatment increased plasma potassium levels and her growth. At the age of one year she was diagnosed as having Bartter's syndrome. Since then she has been treated with indomethacin at an initial dose of 3 mg/kg/day combined with spironolactone and potassium. After the start of the indomethacin treatment, her growth increased dramatically, and her final height was normal adult height. Her puberty developed normally and menarche occurred at the age of 12 years. Levels of serum sodium, chloride, plasma aldosterone and urinary prostaglandin E2 were also normalized. Levels of angiotensin I and II were improved but not within the normal range, but plasma potassium levels slightly decreased after plasma aldosterone levels were normalized and did not change during the treatment period. Plasma renin activity remained high until about the age of 8 years, after which it decreased to almost within the normal range. At 5 months after the start of indomethacin (3 mg/kg/day), her vascular sensitivity to angiotensin II had been improved, and after 2 years and 5 months, her vascular sensitivity was further improved. At this time renin activity had decreased after angiotensin II infusion, but plasma aldosterone did not change. At the age of 16 years (dose of indomethacin: 0.5 mg/kg/day), plasma aldosterone increased after angiotensin II infusion. These data suggest that indomethacin and spironolactone are effective treatments for the neonatal form of Bartter's syndrome, especially during childhood.

Increased expression of angiotensin peptides in the brain of transgenic hypertensive rats.

We determined the levels of angiotensin I (ANG I), angiotensin II (ANG II), and the heptapeptide angiotensin(1-7) [ANG(1-7)] in the blood and brain of female Hannover Sprague-Dawley (SD) and transgenic hypertensive rats [mRen-2]27 by radioimmunoassay and high performance liquid chromatography. Hypertension was accompanied by higher plasma concentrations of ANG II, no statistical changes in ANG(1-7), and no differences in plasma ANG I levels. In the hypothalamus of transgenic rats, concentrations of ANG II and ANG(1-7) averaged 827% and 168% above values in SD rats (p < 0.005) whereas both ANG I and ANG II increased in the medulla oblongata. The data showed that the established phase of hypertension in rats harboring the mouse Ren-2 gene is associated with overexpression of the renin-angiotensin system in brain regions participating in the endocrine regulation of blood pressure.

Nonparallel effects of renin inhibitor treatment on plasma renin activity and angiotensins I and II in hypertensive subjects. An assay-related artifact.

Nonparallel effects of renin inhibitor treatment on plasma renin activity (PRA) and the plasma levels of angiotensins (ANG), as well as on blood pressure, have been observed in subjects with hypertension. This study addresses the possibility that renin inhibitors may show a high degree of plasma protein binding in vivo and that displacement of protein-bound inhibitor during the assay of PRA in vitro may lead to overestimation of renin inhibition. Indeed, with the ultrafiltration technique it was found that 96% of the novel renin inhibitor Ro 42-5892, when added to EDTA plasma, was bound to protein. The angiotensinase inhibitors phenylmethylsulfonyl fluoride (PMSF) and 8-hydroxy-quinoline sulfate (8-OHQ), which are currently used in PRA assays, caused a displacement of protein-bound inhibitor, thereby increasing its free concentration. This displacement was sufficient to explain the reduction in IC 50 of Ro 42-5892, which was seen in the PRA assay when PMSF and 8-OHQ were added to plasma. Such reductions in IC 50 were also seen with the renin inhibitors CGP 29-287, CGP 38-560A, and SR 43-845. When Ro 42-5892 was given, 1 mg/kg intravenously in 10 min, to subjects with hypertension, it appeared that plasma ANG I and II returned to baseline after 6-8 h, whereas PRA measured in the presence of PMSF and 8-OHQ was still suppressed. However, when PRA was measured without these angiotensinase inhibitors, the inhibition of PRA was parallel to the suppression of ANG I and II.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of angiotensin converting enzyme induction and inhibition using quantitative in vitro autoradiography: tissue selective induction after chronic lisinopril treatment.

Angiotensin converting enzyme (ACE) inhibitors lead to induction of ACE in animals and humans. This complicates the use of ACE enzymatic activity as an index of inhibition in plasma or tissues after chronic administration of ACE inhibitors. We have, therefore, developed a method for ACE measurement by in vitro autoradiography using an 125I-labelled inhibitor to quantitate total ACE and the concentration of free (not inhibited) ACE in tissues after prolonged administration of ACE inhibitors to rats. Measurements made on unprocessed tissue sections reflect residual free ACE activity in the presence of the unlabelled inhibitor. In a parallel series of adjacent sections, the ACE inhibitor is dissociated from the enzyme by reversibly denaturing the enzyme by zinc chelation. This is followed by reconstitution of the active enzyme by zinc ion replacement and measuring total enzyme concentration. This technique permits measurement of the extent of ACE inhibition and induction. This method was evaluated in tissues of rats following chronic oral administration of lisinopril (10 mg/kg per day) for 2 weeks. The pattern of ACE inhibition was similar to that seen in our previous acute studies. However, induction of ACE was found to be organ specific; plasma total ACE increased 1.75-fold and total ACE in the lung increased by 30% compared with untreated animals, but there was no demonstrable change in total ACE concentration in the kidney, adrenal or aorta. Despite this, during chronic treatment with lisinopril, ACE activity in all of these organs was inhibited with low levels of free ACE.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-term effects of captopril (SQ14 225) on blood-pressure and hormone levels in essential hypertension.

Captopril, an orally active angiotensin-converting enzyme (ACE) inhibitor, was effective in the long-term reduction of blood-pressure in 17 patients with essential hypertension. The addition of hydrochlorothiazide produced a further hypotensive effect, and the combined treatment produced satisfactory control of the blood-pressure for eight months. Captopril prevented and reversed the secondary hyperaldosteronism and hypokalaemia induced by simultaneous diuretic administration, thus eliminating the need for potassium supplements. The fall in plasma-angiotensin-II and urinary aldosterone and rise in angiotensin I and plasma-renin provide biochemical evidence that captopril inhibits ACE in vivo. No change in circulating venous bradykinin levels could be detected. The hypotensive action of captopril is not mediated by changes in blood-bradykinin but may involve inhibition of the renin-angiotensin and kallikrein-kinin systems locally within the kidneys or blood vessels.