From mitochondria to disease: role of the renin-angiotensin system.

Mitochondria are energy-producing organelles that conduct other key cellular tasks. Thus, mitochondrial damage may impair various aspects of tissue functioning. Mitochondria generate oxygen- and nitrogen-derived oxidants, being themselves major oxidation targets. Dysfunctional mitochondria seem to contribute to the pathophysiology of hypertension, cardiac failure, the metabolic syndrome, obesity, diabetes mellitus, renal disease, atherosclerosis, and aging. Mitochondrial proteins and metabolic intermediates participate in various cellular processes, apart from their well-known roles in energy metabolism. This emphasizes the participation of dysfunctional mitochondria in disease, notwithstanding that most evidences supporting this concept come from animal and cultured-cell studies. Mitochondrial oxidant production is altered by several factors related to vascular pathophysiology. Among these, angiotensin-II stimulates mitochondrial oxidant release leading to energy metabolism depression. By lowering mitochondrial oxidant production, angiotensin-II inhibition enhances energy production and protects mitochondrial structure. This seems to be one of the mechanisms underlying the benefits of angiotensin-II inhibition in hypertension, diabetes, and aging rodent models. If some of these findings can be reproduced in humans, they would provide a new perspective on the implications that RAS-blockade can offer as a therapeutic strategy. This review intends to present available information pointing to mitochondria as targets for therapeutic Ang-II blockade in human renal and CV disease.

Effect of omapatrilat on the aging process of the normal rat.

Previous results have shown that inhibition of the renin-angiotensin system (RAS) either with an angiotensin II (Ang II), type 1 receptor blocker (losartan) or with an angiotensin converting enzyme inhibitor (ACEI, enalapril) has a protective effect on cardiovascular, renal, hepatic and cerebral structure and function during aging. The present study has analyzed the effect of chronic administration of a newly developed compound, omapatrilat, on clinical, histological and biochemical changes due to aging. Omapatrilat combines the action of an ACEI and of an inhibitor of a neutral endopeptidase involved in the metabolism of the atrial natriuretic peptide. The final effect is a decrease of a vasoconstrictor and proinflammatory mechanism like the RAS and the potentiation of two vasodilating compounds like bradykinin and the atrial natriuretic peptide. Based on these actions, its protective effect might be greater than formerly used pharmacological agents. Determinations have been performed on young adults (6 months old), adults (12 months old) or senile (18 months old) rats. Omapatrilat (35 mg/kg/day during 6 months and 20 mg/kg/day thereafter) was administered in the drinking water since weaning until sacrifice. Cardiovascular, renal, and cerebral structure as well as cognitive behavior, cardiovascular and renal function has been analyzed. The biochemical analysis has also established whether the beneficial action of Ang II inhibition is related to an increased activity of the nitric oxide synthase as observed in previous studies. Moreover, this study has tried to determine the relationship between the protective effect of these drugs and the levels of antioxidant defenses present in the blood and/or in the tissues. Hence, enzymatic and non-enzymatic antioxidants have been evaluated.

Angiotensin II blockade improves mitochondrial function in spontaneously hypertensive rats.

Angiotensin II can induce oxidant stress by stimulating vascular superoxide production. Hypertension promotes mitochondrial function decline in brain, liver and heart. The aim of this study was to investigate whether a) hypertension is associated to kidney mitochondrial dysfunction, and b) angiotensin II blockade can reverse potential mitochondrial changes in hypertension. Four-month-old male spontaneously hypertensive rats (SHR) received drinking water containing candesartan (7.5 mg/kg/day, SHR+Cand), or no additions (SHR) for 4-months. Eight-month-old Wistar-Kyoto rats (WKY), that received water with no additions, were used as control. Systolic blood pressure, proteinuria, cortical glomerular area, and glomerular and tubulointerstitial alpha-smooth muscle actin labeling, were significantly higher, and creatinine clearance was significantly lower, in SHR relative to WKY and SHR+Cand. In SHR, kidney mitochondria membrane potential, and nitric oxide synthase and cytochrome oxidase activities were significantly lower than in WKY and SHR+Cand. In SHR, mitochondrial hydrogen peroxide production was significantly higher than in WKY and SHR+Cand. The results suggest that, in hypertension, increased mitochondrial oxidant production may mediate kidney mitochondria dysfunction. Candesartan preserved mitochondrial function, probably favoring the maintenance of adequate cellular and tissue function in the kidney. The known renal protective effects of candesartan in hypertension may be related to the improvement of mitochondrial function. This may be an additional or alternative explanation for some of the beneficial effects of AT1 receptor antagonists.

Candesartan cilexetil protects cavernous tissue in spontaneously hypertensive rats.

In previous experiments, our group demonstrated morphological changes in erectile tissue from male spontaneously hypertensive rats (SHR). The present study was performed to determine whether an angiotensin II receptor blocker could protect cavernous tissue (CT) from these structural alterations in SHR. Male SHR and Wistar-Kyoto (WKY) rats were studied during 4 months. Rats were divided into three groups: SHR (n=10), SHR with candesartan cilexetil (n=10) and WKY rats (n=10). Candesartan cilexetil 7.5 mg/kg/day was administered orally throughout the study. CT was processed for pathology studies. The amount of (1) cavernous smooth muscle (CSM), (2) vascular smooth muscle (VSM), (3) collagen type III, and the rat endothelial cell antibody (RECA-1)/tunica media ratio in cavernous arteries were evaluated. SHR with candesartan cilexetil showed a lower blood pressure, a lower percentage of CSM, smaller VSM area, with a higher RECA-1/media ratio, and a lower percentage of collagen type III, when compared to untreated SHR. In addition, SHR showed a positive correlation between systolic blood pressure (SBP) and CSM amount (r=0.91; P<0.01), and SBP and the percentage of collagen type III (r=0.88; P<0.01); these correlations were not observed either in SHR treated with candesartan cilexetil or in WKY rats. We conclude that candesartan cilexetil provides a significant protective role against morphologic changes in vessels as well as in cavernous spaces of the erectile tissue, caused by high blood pressure, in SHR.

Enalapril attenuates oxidative stress in diabetic rats.

Oxidative stress is involved in both the pathogenesis and complications of diabetes. ACE inhibitors can slow the progression of cardiac and renal impairments related to diabetes. The effect of enalapril treatment on oxidative stress and tissue injury was studied in hearts, kidneys, and livers from streptozotocin-induced diabetic rats. Twenty-four rats were divided into the following groups: streptozotocin (65 mg/kg, single intraperitoneal dose), streptozotocin+enalapril (20 mg enalapril/L drinking water), and control (intraperitoneal saline). Seven months after streptozotocin injection, organs were studied by light microscopy and collagen III immunolabeling. Tissue lesions and collagen labeling were graded by a semiquantitative score (0 to 4). Total glutathione content, glutathione redox status (reduced/oxidized glutathione), antioxidant enzyme activities, protein-associated sulfhydryls, thiobarbituric acid-reactive substances, and fluorescent chromolipids were determined in tissue homogenates. Glycemia was higher in both the streptozotocin and streptozotocin+enalapril groups relative to the control group. In the streptozotocin group, creatinine clearance and body weight were lower, and systolic blood pressure and urinary albumin excretion were higher than in the streptozotocin+enalapril and control groups. Heart, kidney, and liver lesion/labeling scores were significantly higher in the streptozotocin group compared with the streptozotocin+enalapril and control groups. Kidney and liver total glutathione was lower in the streptozotocin group relative to the control group (P<0.05). Enalapril treatment significantly attenuated the reduction of total glutathione. In the heart, kidney, and liver, both glutathione and proteins were relatively more oxidized in the streptozotocin group relative to the control group (P<0.05). Protein and glutathione oxidation were attenuated in the streptozotocin+enalapril group in the 3 tissues studied (P<0.05). Enalapril treatment attenuated the oxidation of lipids in the heart and kidney (P<0.05). Tissue fibrosis scores were inversely correlated with (1) both total glutathione and reduced/oxidized glutathione in heart, kidney, and liver and (2) glutathione reductase activity in the kidney. These results suggest that in streptozotocin-induced diabetic rats, the protective action of enalapril might be mediated, at least in part, by its effect on tissue oxidant/antioxidant status.

Protective role of enalapril for chronic tubulointerstitial lesions of hyperoxaluria.

PURPOSE: Hyperoxaluria is a recognized cause of tubulointerstitial lesions and it may contribute to chronic renal failure. In previous studies we demonstrated that enalapril was effective against the progression of tubulointerstitial lesions in a 4-week hyperoxaluria rat model. We evaluated whether the action of enalapril on the tubulointerstitial lesions produced by hyperoxaluria persisted for a long period. MATERIALS AND METHODS: Two-month-old male Sprague-Dawley rats were divided into 4 groups of 12 each, including 1--control animals given tap water, 2--animals with hyperoxaluria, 3--animals with hyperoxaluria plus enalapril, 4--animals with enalapril. Hyperoxaluria in groups 2 and 3 rats was induced by administering 1% ethylene glycol, a precursor for oxalates, in the tap water continuously throughout the whole study. Meanwhile, groups 3 and 4 received 20 mg./l. enalapril in the drinking water. At the end of the study renal tubulointerstitial lesions were evaluated by immunostaining using monoclonal antibodies against macrophage infiltrates (ED1), tubulointerstitial alpha-smooth muscle actin and transforming growth factor-beta1. The lesions were quantified by semiquantitative scores. Creatinine clearance and urinary albumin excretion were also determined. RESULTS: There was no difference in urine oxalate excretion in groups 2 and 3. Group 3 rats treated with enalapril showed fewer tubulointerstitial lesions than nontreated group 2 rats, as indicated by the mean scores plus or minus standard error of mean for inflammatory infiltrate (2.16 +/- 0.2 versus 0.83 +/- 0.16), tubular atrophy (2 +/- 0.27 versus 0.66 +/- 0.14), interstitial fibrosis (2.5 +/- 0.15 versus 0.5 +/- 0.1), glomerular ED1 (1.75 +/- 0.25 versus 0.16 +/- 0.11), interstitial ED1 (2.33 +/- 0.18 versus 0.58 +/- 0.10) tubular transforming growth factor-beta1 (2.09 +/- 0.08 versus 0.91 +/- 0.14), interstitial transforming growth factor-beta 1 (2.33 +/- 0.22 versus 0.66 +/- 0.12), tubulointerstitial alpha-smooth muscle actin (2.91 +/- 0.22 versus 0.83 +/- 0.16), lower urinary albumin excretion (35.5 +/- 2.7 mg. daily versus 10.9 +/- 1) and higher creatinine clearance (2.29 +/- 0.04 ml. per minute versus 2.54 +/- 0.03, all p <0.05). CONCLUSIONS: Based on our results we believe that enalapril would provide a beneficial effect against chronic tubulointerstitial lesions caused by oxalates.

Effects of dehydroepiandrosterone and quinapril on nephropathy in obese Zucker rats.

BACKGROUND: The obese Zucker rat exhibits insulin resistance, develops nephropathy at an early age, and may be a model of diabetic nephropathy. Dehydroepiandrosterone (DHEA) may ameliorate many of the factors that contribute to diabetic nephropathy, while angiotensin-converting enzyme inhibitors are known to be effective. One marker of nephropathy is the expression of alpha-smooth muscle actin. METHODS: We studied the effect of DHEA on the expression of alpha-smooth muscle actin in obese Zucker rats and compared the changes with those in a control group, a group given quinapril, and a group on a low-calorie diet. DHEA (0.6%) added to plain chow, quinapril (0.3 mg/kg) added to drinking water, and a low-calorie diet based on pair-feeding were administered to obese rats from age 4 to 20 weeks. Immunohistochemical expression of alpha-smooth muscle actin, a marker of interstitial and glomerular fibrosis and an early indicator of nephropathy, was measured semiquantitatively in glomeruli, cortical interstitium, and medullary interstitium on a scale of 0 to 4 and was reported as mean +/- SEM. RESULTS: When compared with the obese control group, quinapril exhibited a marked reduction in alpha-smooth muscle actin staining in glomeruli, cortical interstitium, and medullary interstitium (P < 0.0005); DHEA reduced alpha-smooth muscle actin staining in cortical interstitium and medullary interstitium (P < 0. 005), and a low-calorie diet reduced alpha-smooth muscle actin staining in cortical and medullary interstitium (P < 0.005), which was similar to the effects of DHEA. CONCLUSIONS: DHEA was similar to a low-calorie diet in reducing the immunohistochemical staining of alpha-smooth muscle actin in obese Zucker rats. However, quinapril exerted a marked protective effect on the development of fibrosis, as indicated by alpha-smooth muscle actin staining, which was significantly less than that of DHEA at the doses studied.

Morphological changes in cavernous tissue in spontaneously hypertensive rats.

Erectile dysfunction has an increased prevalence in hypertensive patients and is associated with cardiovascular diseases. For many years the discussion has been polarized on whether in hypertensive patients, it is the arterial hypertension or the antihypertensive therapy that is the cause of male erectile dysfunction. The aim of our study was to determine the morphologic changes in cavernous tissue (CT) in an animal model of arterial hypertension. Male spontaneously hypertensive rats (SHR) (n = 15) and normotensive Wistar-Kyoto (WKY) rats (n = 15) were studied for 8 months. Animals were allowed to drink tap water and fed a standard rat chow ad libitum. Systolic blood pressure (SBP) was measured monthly by the tail/cuff method. At the end of the experiment all the animals were sacrificed for microscopic studies. Cavernous tissue was processed by hematoxylin and eosin, Masson's trichrome, and monoclonal anti-alpha smooth muscle actin. Cavernous smooth muscle (CSM) and vascular smooth muscle (VSM) proliferation and CT fibrosis were evaluated by a semiquantitative score. SHR showed a higher proliferative score in CSM (2.7 +/- 0.28 v 1.1 +/- 0.07; P < .001), as well as in VSM (2.7 +/- 0.25 v 1 +/- 0.05; P < .001), and higher CT fibrosis score (2.8 +/- 0.28 v 0.1 +/- 0.07; P < .001), when compared to WKY rats. Furthermore, SHR showed a positive correlation between SBP and CSM proliferative score (r2 = 0.9277), SBP and VSM proliferative score (r2 = 0.8828), and SBP and CT fibrosis score (r2 = 0.7775). In addition, an increase in the surrounding connective tissue at the perineurium and endoneurium of the amielinic nerves in CT was observed in the SHR group. According to these results we conclude that SHR present morphologic changes in vessels as well as in cavernous spaces of the erectile tissue that have a high positive correlation with high blood pressure. Moreover, the increase in extracellular matrix expansion seems to affect not only the interstitium but also the neural structures of the penis.

Enalapril and captopril enhance glutathione-dependent antioxidant defenses in mouse tissues.

The effect of enalapril and captopril on total glutathione content (GSSG + GSH) and selenium-dependent glutathione peroxidase (Se-GPx) and glutathione reductase (GSSG-Rd) activities was investigated in mouse tissues. CF-1 mice (4-mo-old females) received water containing enalapril (20 mg/l) or captopril (50 mg/l) for 11 wk. Enalapril increased GSSG + GSH content (P < 0.05) in erythrocytes (147%), brain (112%), and lung (67%), and captopril increased GSSG + GSH content in erythrocytes (190%) and brain (132%). Enalapril enhanced Se-GPx activity in kidney cortex (42%) and kidney medulla (23%) and captopril in kidney cortex (30%). GSSG-Rd activity was enhanced by enalapril in erythrocytes (21%), brain (21%), liver (18%), and kidney cortex (53%) and by captopril in erythrocytes (25%), brain (19%), and liver (34%). In vitro erythrocyte oxidant stress was evaluated by thiobarbituric acid-reactive substances (TBARS) production (control 365 +/- 11, enalapril 221 +/- 26, captopril 206 +/- 17 nmol TBARS x g Hb(-1) x h(-1); both P < 0.05 vs. control) and phenylhydrazine-induced methemoglobin (MetHb) formation (control 66.5 +/- 3.5, enalapril 52.9 +/- 0.4, captopril: 56.4 +/- 2.9 micromol MetHb/g Hb; both P < 0.05 vs. control). Both angiotensin-converting enzyme inhibitor treatments were associated with increased nitric oxide production, as assessed by plasma NO-(3) + NO-(2) level determination (control 9.22 +/- 0.64, enalapril 13.7 +/- 1.9, captopril 17.3 +/- 3.0 micromol NO-(3) + NO-(2)/l plasma; both P < 0.05 vs. control). These findings support our previous reports on the enalapril- and captopril-induced enhancement of endogenous antioxidant defenses and include new data on glutathione-dependent defenses, thus furthering current knowledge on the association of ACE inhibition and antioxidants.

Effects of amlodipine on tubulointerstitial lesions in normotensive hyperoxaluric rats.

Although controversial, a number of reports have suggested that calcium antagonists can retard or prevent the progression of various renal diseases in experimental models. Nevertheless, there are few data related to tubulointerstitial changes in these studies. On the other hand, hyperoxaluria is a recognized cause of tubulointerstitial lesions, and this could contribute to the development of hypertension and chronic renal failure. The aim of the present study was to evaluate a possible beneficial effect of amlodipine, a 1,4-dihydropyridine class of calcium antagonist, in a model of primary tubulointerstitial lesion produced by hyperoxaluria. Two-month-old male Sprague-Dawley rats were separated into 4 groups for a 4-week period: G1 (control; tap water only); G2 (hyperoxaluric); G3 (hyperoxaluric plus amlodipine treatment); and G4 (amlodipine treatment). G2 and G3 rats were given 1% ethylene glycol (a precursor for oxalates) in drinking water, and G3 and G4 rats were given amlodipine 2 mg. kg(-1). d(-1) by gavage. At the end of the study, we evaluated by semiquantitative scores (0 to 4) the different renal tubulointerstitial lesions, urinary albumin excretion, renal function by creatinine clearance, and blood pressure. Rats belonging to the hyperoxaluric group treated with amlodipine (G3) had fewer tubulointerstitial lesions, as follows: (1) inflammatory infiltrate score: 3.31+/-0.07 versus 0.23+/-0.12; P<0.05; (2) tubular atrophy score: 3.33+/-0.33 versus 0.50+/-0.22, P<0.05; (3) interstitial fibrosis score: 2.76+/-0.34 versus 0.31+/-0. 16, P<0.05; (4) oxalate deposits score: 3.66+/-0.33 versus 0.09+/-0. 08, P<0.05; (5) lower urinary albumin excretion (11.3+/-2 versus 27+/-4.5 mg/d, P<0.01); and (6) higher creatinine clearance (1. 22+/-0.08 versus 1.13+/-0.08, P<0.01) compared with the hyperoxaluric group untreated with amlodipine (G2). On the other hand, there were no significant changes in blood pressure in any group. In view of these data, we suggest that amlodipine, probably by nonhemodynamic mechanisms of action, can provide an important benefit in the prevention of epithelial tubular cell injury and inflammatory response and therefore in the prevention of the progressive tubulointerstitial fibrosis caused by oxalates.

Higher levels of antioxidant defenses in enalapril-treated versus non-enalapril-treated hemodialysis patients.

We previously reported chronic treatment with angiotensin-converting enzyme inhibitors (ACEis) increases antioxidant defenses in mice. In the present study, however, we examined various antioxidant defenses in chronic hemodialysis (HD) patients either treated with enalapril (10 mg/d) for at least 6 months (+ACEi; n = 11) or untreated (-ACEi; n = 11). The relationship between antioxidant status and HD was investigated by determining oxidative stress markers and antioxidant defenses in a group of chronic HD patients (n = 33) and a group of age-matched controls (n = 29). The effect of a single HD session on those parameters was also evaluated. Before an HD session (pre-HD), HD patients had significantly lower levels of red blood cell (RBC) glutathione (GSH), selenium-dependent glutathione peroxidase activity (RBC-Se-GPx), plasma ubiquinol-10, and alpha-tocopherol than controls. In a randomly selected group of patients (n = 19), a single HD session caused an additional decrease in RBC-GSH and plasma ubiquinol-10 levels. Plasma thiobarbituric acid reactive substance (TBARS) levels were significantly greater in pre-HD patients than controls. Post-HD plasma TBARS levels were similar to control values. The cohort of +ACEi HD patients had greater pre-HD RBC-GSH content, RBC-Se-GPx activity, and plasma beta-carotene concentrations than -ACEi patients (RBC-GSH: +ACEi, 3.1 +/- 0.9 micromol/mL packed RBCs [PRBCs]; -ACEi, 1.2 +/- 0.3 micromol/mL PRBCs [P < 0.05 v +ACEi]; RBC-Se-GPx: +ACEi, 5.8 +/- 0.7 U/mL PRBCs; -ACEi, 4.3 +/- 0.2 U/mL PRBCs [P < 0.05 v +ACEi]; plasma beta-carotene: +ACEi, 0.54 +/- 0.16 micromol/L plasma; -ACEi, 0.19 +/- 0.05 micromol/L plasma [P < 0.05 v +ACEi]). Results show profound alterations in the circulating antioxidant systems of chronic HD patients and that additional oxidative stress occurs during the HD procedure. In addition, in +ACEi HD patients, the levels of several antioxidant defenses are greater than in those in -ACEi HD patients.

Enalapril prevents tubulointerstitial lesions by hyperoxaluria.

Hyperoxaluria is a recognized cause of tubulointerstitial lesions, and this could contribute to development of hypertension and chronic renal failure. Enalapril has been effective against the progression of tubulointerstitial lesions in various animal models. The aim of the present study was to evaluate the usefulness of enalapril on the tubulointerstitial damage produced by oxalates. Two-month-old male Sprague-Dawley rats were separated into 4 groups, control with tap water (G1), hyperoxaluric (G2), hyperoxaluric+enalapril (G3), enalapril (G4), for 4 weeks. G2 and G3 rats were given 1% ethyleneglycol (ETG, precursor for oxalates), and G3 and G4 rats were given enalapril 20 mg/L in drinking water. At the end of the study, we evaluated renal tubulointerstitial lesions by a semiquantitative score. Urine albumin excretion, serum and urine nitric oxide production, tubulointerstitial immunostaining by alpha-smooth muscle actin, transforming growth factor-beta1, and collagen type III were measured. Rats belonging to the hyperoxaluric group treated with enalapril (G3) showed fewer tubulointerstitial lesions (1.3+/-0.2 versus 3+/-0.2; P<0.01), lower urine albumin excretion (8+/-2 mg/d versus 25+/-2 mg/d; P<0.01), less percentage of alpha-smooth muscle actin in renal interstitium (2+/-0.4% versus 13.5+/-2.4%; P<0.01), less percentage of transforming growth factor-beta1 in tubulointerstitial area (3.3+/-1% versus 13.3+/-2. 1%; P<0.01), less percentage of collagen type III interstitial deposition (0.7+/-0.5% versus 7+/-2.6%; P<0.01), and increased NO production in serum as well as urine (both P<0.01), when compared with the hyperoxaluric group not treated with enalapril (G2). Considering these data, we believe that enalapril, by several mechanisms of action, could provide an important benefit in the prevention of inflammatory response, transforming growth factor-beta1 tubulointerstitial production, collagen type III interstitial deposition, and finally, the progressive tubulointerstitial fibrosis caused by oxalates.

Biomolecular changes in the aging myocardium: the effect of enalapril.

Chronic administration of enalapril in the aging mouse prevents myocardial fibrosis. To investigate the mechanisms involved, we studied 30 CF1 female mice that received enalapril (ENAL:20 mg/L) in their drinking water after weaning and 30 control (CONT) mice. Ten animals from each group were killed at 12, 18, and 24 months. Half of the samples were prepared for light microscopy (LM) and the other half for electron microscopy (EM). Cardiac histologic sections were studied by an image analyzer (Bioscan OPTIMAS 4.1). We performed the following measurements in cardiomyocytes: mitochondrial number, mitochondrial superoxide dismutase (SOD) using immunohistochemical methods with EM, the percentage of cell cyclin, and apoptosis. The results obtained for CONT and ENAL, respectively were as follows. For cyclin (percentage of positive) our results were: 12 months 17.1+/-0.1% and 18.2+/-0.8%, 18 months 2.4+/-1.6% (P < .001), and 11.4+/-0.1% (P < .001), 24 months 1.2+/-1.3% (P < .001), and 8.2+/-1.2% (P < .001) with significant differences at 18 and 24 months. For the Feulgen method (cell/mm2) we found: 12 months CONT 89.7+/-1.2, ENAL 84.6+/-1.2; 18 months CONT 62.8+/-1.2, ENAL 98.7+/-1.3, and 24 months CONT 81.2+/-1.3, ENAL 112.3+/-1.4. Apoptosis (percentage of positive) was found to be 12 months 3.7+/-0.4% and 1.9+/-0.1%, 18 months 7.1 +/-0.3% (P < .001), and 1.5+/-0.1% (P < .001), 24 months 10.9+/-0.5% (P < .001) and 2.1+/-1.8% (P < .001), for CONT and ENAL, respectively; there were significant differences at 18 and 24 months. The number of mitochondria per cardiomyocyte were: 12 months 85.9+/-1.8 and 87.3+/-1.5, 18 months 69.2+/-1.5t and 82.2+/-1.8 (P < .001), 24 months 54.6+/-1.1 (P < .001) and 81.4+/-1.6 (P < .001) for CONT and ENAL respectively, with significant differences at 18 and 24 months. Mitochondrial SOD was found to be: 12 months 13.6%+/-0.2% (P < .05) and 17.8%+/-1.3% (P < .05), 18 months 7.1%+/-1.0% (P < .001) and 16.7%+/-1.6% (P < .001), 24 months 4.1%+/-0.5% (P < .001), and 12.4%+/-0.9% (P .001) for CONT and ENAL respectively, with significant differences at 12 months and at 18 and 24 months (ANOVA and contrast Scheffe's test). We conclude that chronic administration of ENAL modifies mitochondrial SOD at 12 months, whereas at 18 and 24 months ENAL was associated with higher mitochondrial SOD and a higher mitochondrial number with a greater cyclin expression, and a lower percentage of apoptosis. Enalapril may prevent myocardial fibrosis, possibly by causing changes related to enzymatic-mitochondrial or cellular cycle modifications.

Enalapril and captopril enhance antioxidant defenses in mouse tissues.

This study was conducted to investigate a possible systemic effect of angiotensin-converting enzyme inhibitors (ACEi) on tissue antioxidant defenses. CF1 mice (4-mo-old females) were administered either water (control) or water containing enalapril (20 mg/l) or captopril (50 mg/l) during 11 wk. Neither enalapril nor captopril treatment had an effect on body mass or brain, kidney, or heart weight relative to controls. CuZn-superoxide dismutase (SOD) activity was increased by enalapril treatment in kidney medulla (27%), heart (24%), and erythrocytes (19%) and by captopril treatment in kidney medulla (43%) and heart (54%) relative to controls. Mn-SOD and catalase activities were unaffected by either treatment. Enalapril, but not captopril treatment, increased Se-glutathione peroxidase activity in renal medulla (19%). Nonenzymatic antioxidant defenses, evaluated by tert-butyl hydroperoxide-initiated chemiluminescence (HICL), were enhanced in kidney cortex (48%) by enalapril and in brain by enalapril (44%) or captopril (36%) treatment relative to controls. As evaluated in vitro by HICL and thiobarbituric acid-reactive substances formation, captopril had a free radical scavenger activity, whereas neither enalapril nor lisinopril was effective. These results suggest that ACEi may protect tissues from oxidative damage by increasing enzymatic and nonenzymatic antioxidant defenses.

Renal interstitial sclerosis in aging: effects of enalapril and nifedipine.

The effects of nifedipine and enalapril on age-associated renal interstitial fibrosis were investigated in 60 CF1 female mice. Mice received 20 mg enalapril (ENAL) per L (N = 20), or 40 mg nifedipine (NIF) per L (N = 20) in their drinking water. Control (CONT) mice received tap water ad libitum. The percentages of both interstitial peritubular sclerosis (IPS) in cortex and interstitial medullary sclerosis (IMS) were determined. Kidney tissue was studied using immunological techniques and optical (OM) and electron microscopy (EM) to analyze the expression of renin. alpha-SM-actin and vimentine expression were also evaluated. The results showed that blood pressure levels in ENAL or NIF animals were not different from those of CONT. Renin expression was observed in arcuate vessels (AV) in ENAL animals, whereas no renin staining in AV was found in either NIF or CONT animals. Renin immunoreactivity in the juxtaglomerular apparatus was more intense in ENAL mice, as compared with NIF or CONT animals. Laboratory testing showed the following values: proteinuria (mg/mL): CONT 6.1 +/- 0.6, NIF 11.2 +/- 2.3, and ENAL 1.0 +/- 0.6 (P < 0.05); creatinine: CONT 1.37 +/- 0.24, NIF 0.87 +/- 0.16, and ENAL 0.63 +/- 0.1 (P < 0.01). The percentages of interstitial sclerosis were: %IPS: CONT 18.12 +/- 1.1, NIF 17.40 +/- 0.9, and ENAL 3.42 +/- 1.3 (P < 0.01); %IMS: CONT 23.41 +/- 1.5, NIF 21.80 +/- 1.9, and ENAL 6.12 +/- 1.2 (P < 0.01). Percentages of alpha-SM-actin expression were: CONT 13.10 +/- 1.9, NIF 13.80 +/- 0.2, and ENAL 1.00 +/- 0.1 (P < 0.01). Vimentine staining showed no differences among the groups. It was concluded that enalapril reduces the peritubular and medullar interstitial fibrosis, whereas nifedipine has no effect.

Decrease of exercise-induced microalbuminuria in patients with type I diabetes by means of an angiotensin-converting enzyme inhibitor.

Taking into account both the importance of microalbuminuria (MA) as a predictive parameter of clinical nephropathy in diabetic patients and the efficiency of exertion to show and/or to increase MA in both diabetic patients and normal individuals, we studied 37 type I diabetic patients divided into two groups: group A, with no MA at rest (n = 19), and group B, with MA at rest (n = 18). Group C comprised 10 healthy volunteers as controls. Changes of basal MA during exercise and postexercise were studied in all three groups. Normotensive patients with no metabolic disorders, normal renal function, and no proteinuria underwent an ergometric test up to 600 kg. This test was repeated after the administration of 20 mg enalapril in a single daily dose for 60 days. Body weight, systolic and diastolic arterial pressure, creatinine, and creatinine clearance were determined and showed no significant variations either between groups or with treatment. Microalbuminuria was studied in the three groups with and without administration of enalapril throughout the 2 months of the study. Determinations were performed under conditions of rest, exercise, and postexercise. Mean baseline MA values +/- SEM were as follows: at rest, 5.22 +/- 0.49, 58.36 +/- 13.24, and 4.73 +/- 0.45 micrograms/min for groups A, B, and C, respectively; with exercise, 15.19 +/- 4.43, 74.70 +/- 14.89, and 16.76 +/- 4.62 micrograms/min for groups A, B, and C, respectively; and postexercise, 32.04 +/- 6.64, 253.15 +/- 63.88, and 9.23 +/- 3.25 micrograms/min, respectively. The geometric means of the baseline to posttreatment MA ratio were as follows: at rest, 0.95, 1.59 (P < 0.01), and 1.03 for groups A, B, and C, respectively; with exercise, 1.53 (P < 0.01), 1.91 (P < 0.01), and 1.69 for groups A, B, and C, respectively; and postexercise, 2.94 (P < 0.01), 3.24 (P < 0.01), and 1.03 for groups A, B, and C, respectively. In conclusion, in the early diagnostic suspicion of diabetic nephropathy, the screening of postexercise MA during an ergometric test could be of help. Treatment with enalapril decreased MA in diabetic groups A (no MA at rest) and B (MA at rest) during exercise and postexercise, and also decreased MA in group B while at rest.

Superoxide dismutase and glutathione peroxidase activities are increased by enalapril and captopril in mouse liver.

We have characterized the effect of angiotensin converting enzyme (ACE) inhibitors on the activity of CuZn-superoxide dismutase (CuZn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase, and selenium-dependent glutathione peroxidase (Se-GPx). CF1 mice (4-month-old females) were administered water containing enalapril (20 mg/l) or captopril (50 mg/l), during 4 to 11 weeks. After 11 weeks, enalapril treatment caused an increase in the activity of CuZn-SOD, Mn-SOD and Se-GPx, from 19 +/- 4 to 46 +/- 7, 2.1 +/- 0.2 to 3.8 +/- 0.2 units/mg protein and 27 +/- 3 to 54 +/- 3 milliunits/mg protein, respectively. After 11 weeks, captopril treatment increased the activities (P < 0.05) of CuZn-SOD, MnSOD and Se-GPx to 35 +/- 4, 2.9 +/- 0.2 units/mg protein, and 38 +/- 2 milliunits/mg protein, respectively. Catalase activity was not affected by the treatments. These results suggest that ACE inhibitors may protect cell components from oxidative damage by increasing the enzymatic antioxidant defenses.

Cardiovascular changes by long-term inhibition of the renin-angiotensin system in aging.

We studied four groups of 20 female mice to evaluate the long-term effect of an angiotensin-converting enzyme on myocardium and vessels during the natural process of aging. Three groups received enalapril in water from weaning to 24 months of age (group A, 20 mg/L; group B, 10 mg/L; group C, 5 mg/L); group D served as a control. Animals surviving after 24 months were killed, and morphometric studies were performed. Total corporal weight was higher in animals receiving enalapril. Cardiac weight relative to total body weight was lower in the treated groups than in the control group. Cardiac morphometric studies showed lower myocardiosclerosis in animals receiving angiotensin-converting enzyme inhibitor (groups A through D, respectively, 0.9 +/- 0.6%, 1.1 +/- 0.2%, 1.03 +/- 0.1%, and 9.5 +/- 4.3%; P < .01, groups A, B, and C versus D). The number of mitochondria per myocardiocyte was higher in the groups receiving enalapril (A through D, respectively, 85 +/- 7, 85 +/- 6, 83 +/- 8, and 58 +/- 8; P < .01, groups A, B, and C versus D). At the vascular level, vessel diameters were not significantly different between the groups receiving angiotensin-converting enzyme inhibitor and the control group, whereas differences were seen in arterial tunica media thickness (wall-lumen ratio) (groups A through D, respectively, aorta: 0.13 +/- 0.02, 0.11 +/- 0.02, 0.12 +/- 0.01, 2.81 +/- 0.35; intrapulmonary: 0.9 +/- 0.43, 0.6 +/- 0.41, 0.8 +/- 0.46, 1.9 +/- 0.51; intracerebral: 2.18 +/- 0.46, 2.29 +/- 0.45, 2.46 +/- 0.43, 3.30 +/- 0.41; intrarenal: 2.28 +/- 0.46, 2.73 +/- 0.48, 2.70 +/- 0.51, 3.23 +/- 0.41; intracariaciac: 2.27 +/- 0.44, 2.59 +/- 0.41, 2.80 +/- 0.43, 3.68 +/- 0.47; P < .001, groups A, B, and C versus D).(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased glomerulosclerosis in aging by angiotensin-converting enzyme inhibitors.

To evaluate the effects of angiotensin-converting enzyme inhibition on renal aging, enalapril was administered in the drinking water to three groups of CF1 mice at doses of 20 mg/L (Group A), 10 mg/L (Group B), and 5 mg/L (Group C). These experimental groups were compared with 20 CF1 mice not receiving enalapril (Group D). At 2 yr, total body weight was 48.1 +/- 7.5 g in Group A, 47.7 +/- 7.1 g in Group B, 47.6 +/- 4.6 g in Group C, and 35.1 +/- 5.4 g in Group D. The ratio of kidney to total body weight, in percentages, was 1.8 +/- 0.3, 1.6 +/- 0.3, 1.9 +/- 0.2, and 1.5 +/- 0.1 in Groups A, B, C, and D, respectively. Morphometric studies of the kidneys revealed the glomerular diameter to be 86.7 +/- 18.0 microns, 96.9 +/- 6.3 microns, 91.1 +/- 11.4 microns, and 106.8 +/- 9.3 microns in Groups A, B, C, and D, respectively. The number of glomeruli per square millimeter of renal cortex was 9.6 +/- 3.7, 12.3 +/- 2.7, 12.4 +/- 8.6, and 3.2 +/- 1.5 in Groups A, B, C, and D, respectively. The mesangial area per glomerulus, in percentages, was 11.6 +/- 4.8, 13.9 +/- 2.9, 14.2 +/- 3.1, and 20.6 +/- 1.9 in Groups A, B, C, and D, respectively. The percentage of glomeruli with sclerosis was 0.1 +/- 0.1, 0.3 +/- 0.1, 0.6 +/- 0.2, and 11.6 +/- 1.9 in Groups A, B, C, and D, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)