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.

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.

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.

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.

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 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.

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)

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.

[Renin synthesis by renal cells during chronic inhibition of angiotensin I converting enzyme]. / Síntesis de renina por células renales durante la inhibición crónica de la enzima convertidora de la angiotensina I.

A study was performed on renin synthesis in order to evaluate changes that occur in renal cells during angiotensin-converting enzyme chronic inhibition of Ang I in mice. Immediately after weaning, 20 CF1 mice received 20 mg/l enalapril maleate in drinking water during 16 months; this group was compared with a control group. Kidney tissue was processed and studies using optical and electron microscope immunochemical techniques were performed. An antirenin antibody was used, and in situ hybridization was performed to keep track of renin mRNA with a digoxygenin-marked probe. We calculated the number of juxtaglomerular apparatus (JGA), afferent arterioles (AA) and arcuate vessels (AV) immunomarked (IM) with antirenin and antidigoxygenin. These parameters were rendered in JGA rates (%IMJGA) and AA (%IMAA) and AV (%IMAV) marked rates (%AV), and in the rate of JGA (%SJGA), AA (%SAA) and AV (%SAV) hybridization signs. Electron microscope readings were used to determine the number of gold particles per renin granule. An increase in the number of renin-producing cells was observed in animals having received enalapril chronically, beyond AJG and AA, since marking was observed in arcuate vessels. The mean %MJGA value was lower in control animals (65.6% +/- 2.4) than in treated animals (94.2% +/- 3 p < 0.05). Similar findings occurred with %MAA: 23.6% +/- 3, (control animals) vs. 41.6% +/- 2.3, p < 0.05 (treated animals). AV were not marked in the control group, as they were in treated animals where %MAV was 4.4% +/- 1.6. The mRNA distribution was different in animals with RAS inhibition as compared with control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Síntesis de renina por células renales durante la inhibición crónica de la enzima convertidora de la angiotensina I. / [Renin synthesis by renal cells during chronic inhibition of angiotensin I converting enzyme]

A study was performed on renin synthesis in order to evaluate changes that occur in renal cells during angiotensin-converting enzyme chronic inhibition of Ang I in mice. Immediately after weaning, 20 CF1 mice received 20 mg/l enalapril maleate in drinking water during 16 months; this group was compared with a control group. Kidney tissue was processed and studies using optical and electron microscope immunochemical techniques were performed. An antirenin antibody was used, and in situ hybridization was performed to keep track of renin mRNA with a digoxygenin-marked probe. We calculated the number of juxtaglomerular apparatus (JGA), afferent arterioles (AA) and arcuate vessels (AV) immunomarked (IM) with antirenin and antidigoxygenin. These parameters were rendered in JGA rates (

IMJGA) and AA (

IMAA) and AV (

IMAV) marked rates (

AV), and in the rate of JGA (

SJGA), AA (

SAA) and AV (

SAV) hybridization signs. Electron microscope readings were used to determine the number of gold particles per renin granule. An increase in the number of renin-producing cells was observed in animals having received enalapril chronically, beyond AJG and AA, since marking was observed in arcuate vessels. The mean

MJGA value was lower in control animals (65.6

+/- 2.4) than in treated animals (94.2

+/- 3 p < 0.05). Similar findings occurred with

MAA: 23.6

+/- 3, (control animals) vs. 41.6

+/- 2.3, p < 0.05 (treated animals). AV were not marked in the control group, as they were in treated animals where

MAV was 4.4

+/- 1.6. The mRNA distribution was different in animals with RAS inhibition as compared with control animals.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo incorporation of [U-(14)C]glycerol and [2-(3)H]glycerol into phospholipids of brain subcellular fractions in normal and malnourished animals.

A double label design was used to study the in vivo incorporation of [U-(14)C] and [2-(3)H]glycerol into total and individual phospholipids of various brain subcellular fractions isolated from 20-day old normal and undernourished rats. In control animals, synthesis of glycerophospholipids of microsomes, mitochondria and nerve endings seems to occur through the glycerol-3-phosphate (G-3-P) pathway while a large part of the synthesis of myelin glycerophospholipids appears to proceed through the dihydroxyacetone phosphate (DHAP) pathway. In starved animals, on the other hand the incorporation of phospholipid precursors through the DHAP pathway was found to be lower than in controls while synthesis of phospholipids in the other subcellular fractions was unaffected. The possible relationship between the synthesis of glycerophospholipids and especially plasmalogens of the myelin membrane and microperoxisomes of oligodendroglial cells, where the enzymes of the DHAP pathway are located, is discussed.