Elena M.V. de Cavanagh

Enalapril and losartan attenuate mitochondrial dysfunction in aged rats

Renin‐angiotensin system (RAS) inhibition can attenuate the effects of aging on renal function and structure; however, its effect on mitochondrial aging is unknown. To investigate whether an angiotensin‐converting enzyme inhibitor (enalapril) or an angiotensin II receptor blocker (losartan) could mitigate age‐associated changes in kidney mitochondria, male Wistar rats (14 mo old) received during 8 mo water containing either enalapril (10 mg/kg/day) (Enal), or losartan (30 mg/kg/day) (Los), or no additions (Old). Four‐month‐old untreated rats (Young) were also studied. In Old rats mitochondrial respiratory control, ADP/O, nitric oxide synthase activity, and uncoupling protein 2 levels were lower (46, 42, 27, and 76%, respectively), and Mn‐SOD activity was higher (70%) than in Young, Enal, and Los rats. In Old rats mitochondrial hydrogen peroxide production was higher than in both Young (197%) and Enal or Los (40%) rats. In Old rats, kidney GSH/GSSG was lower than in both Young (80%) and Enal (57%) or Los (68%) rats. In Old rats electron microscopy showed effacement of microvilli in tubular epithelial cells, ill‐defined mitochondrial cristae, lower mitochondrial numbers, and enhanced number of osmiophilic bodies relative to Young, Enal, or Los rats. In conclusion, enalapril and losartan can protect against both age‐related mitochondrial dysfunction and ultrastructural alterations, underscoring the role of RAS in the aging process. An association with oxidative stress modulation is suggested.

Protective effect of the inhibition of the renin-angiotensin system on aging.

Experimental studies indicate that chronic long-term inhibition of the renin-angiotensin system (RAS) can prevent most of the deleterious effects due to aging in the cardiovascular system and in the kidney of the normal mouse and rat. In this review, all the information available on this subject provided by several studies performed by our research group during the last years is been described. Treatment was initiated either after weaning or at 12 months of age that is about half the normal life span of the rat. A converting enzyme inhibitor: enalapril or an angiotensin II type 1 (AT1) receptor blocker: losartan were used to inhibit the RAS. Cognitive behaviour, emotionality, and locomotor activity were also determined at 10 and 18 months of age in treated since weaning and untreated control rats to elucidate the participation of angiotensin II in memory disfunction. A similar observation was obtained in animals treated from 12 to 18 months of age. Results have demonstrated a significant protective effect on the function and the structure of the cardiovascular system, the kidney and the brain in all the treated animals. Damage observed at 12 months of age was not very significant, but treatment stop further deterioration that was evident in untreated animals. The similarity of the results detected with either enalapril or losartan treatment, clearly indicates that most of the effects are exerted through AT1 receptors. Analysis of the nitric oxide and antioxidant enzymes systems suggest that the protective effect is related to an antioxidant action of the RAS inhibitors and a reduced formation of reactive oxygen species. AngII inhibition might produce changes in the mechanisms of oxidative stress specially at the mitochondrial level. Prevention of mitochondrial decrease and/or damage would be related with the delay of the normal aging process.

Angiotensin II, mitochondria, cytoskeletal, and extracellular matrix connections: an integrating viewpoint

Malfunctioning mitochondria strongly participate in the pathogenesis of cardiovascular damage associated with hypertension and other disease conditions. Eukaryotic cells move, assume their shape, resist mechanical stress, accommodate their internal constituents, and transmit signals by relying on the constant remodeling of cytoskeleton filaments. Mitochondrial ATP is needed to support cytoskeletal dynamics. Conversely, mitochondria need to interact with cytoskeletal elements to achieve normal motility, morphology, localization, and function. Extracellular matrix (ECM) quantity and quality influence cellular growth, differentiation, morphology, survival, and mobility. Mitochondria can sense ECM composition changes, and changes in mitochondrial functioning modify the ECM. Maladaptive ECM and cytoskeletal alterations occur in a number of cardiac conditions and in most types of glomerulosclerosis, leading to cardiovascular and renal fibrosis, respectively. Angiotensin II (ANG II), a vasoactive peptide and growth factor, stimulates cytosolic and mitochondrial oxidant production, eventually leading to mitochondrial dysfunction. Also, by inducing integrin/focal adhesion changes, ANG II regulates ECM and cytoskeletal composition and organization and, accordingly, contributes to the pathogenesis of cardiovascular remodeling. ANG II-initiated integrin signaling results in the release of transforming growth factor-beta(1) (TGF-beta(1)), a cytokine that modifies ECM composition and structure, induces reorganization of the cytoskeleton, and modifies mitochondrial function. Therefore, it is possible to hypothesize that the depression of mitochondrial energy metabolism brought about by ANG II is preceded by ANG II-induced integrin signaling and the consequent derangement of the cytoskeletal filament network and/or ECM organization. ANG II-dependent TGF-beta(1) release is a potential link between ANG II, ECM, and cytoskeleton derangements and mitochondrial dysfunction. It is necessary to emphasize that the present hypothesis is among many other plausible explanations for ANG II-mediated mitochondrial dysfunction. A potential limitation of this proposal is that the results compiled here were obtained in different cells, tissues, and/or experimental models.

Effects of angiotensin II subtype 1 receptor blockade by losartan on tubulointerstitial lesions caused by hyperoxaluria.

PURPOSE Hyperoxaluria is a recognized cause of tubulointerstitial lesions and this circumstance could contribute to cause chronic renal disease. The renin-angiotensin system has a critical role in the development of interstitial fibrosis, mostly by angiotensin II type 1 receptor stimulation of pro-fibrotic mechanisms. We evaluated whether angiotensin II type 1 receptor blockade prevents oxalate renal lesions. MATERIALS AND METHODS We divided 2-month-old male Sprague-Dawley rats into 4 groups, namely group 1-control, group 2-hyperoxaluria, group 3-hyperoxaluria plus losartan and group 4-losartan. For 4 weeks groups 2 and 3 received 1% ethylene glycol (precursor for oxalates) in drinking water. Losartan (40 mg./kg. body weight) was administered in groups 3 and 4 daily. At the end of the study renal lesions were evaluated using anti-alpha-smooth muscle actin, anti-collagen type III, anti-monocytes/macrophages and anti-transforming growth factor-beta1 antibodies. To evaluate oxidative stress in renal tissue total glutathione and thiobarbituric acid reactive substances in kidney homogenates were determined. Regarding renal functional parameters, creatinine clearance and urinary albumin excretion were also studied. RESULTS Despite similar urinary oxalate levels compared with group 2 group 3 rats showed fewer tubulointerstitial lesions, consisting of significant lower scores for tubular atrophy, unspecific inflammatory cell infiltrate, ED1 mouse anti-rat monoclonal antibody (Serotec, Ltd., Oxford, United Kingdom) (monocytes/macrophages), crystal deposits, interstitial fibrosis, alpha-smooth muscle actin, collagen type III and tubulointerstitial transforming growth factor-beta1. Moreover, urinary albumin excretion and creatinine clearance were significantly improved in group 3 (p <0.01). Higher total glutathione and lower thiobarbituric acid reactive substances were also observed in this group (p <0.01). Thiobarbituric acid reactive substances were the most important and significant independent variable correlating with interstitial fibrosis (t ratio 4.867, p <0.04). CONCLUSIONS We believe that the renal-angiotensin system interaction by losartan produces a beneficial effect against renal lesions caused by hyperoxaluria through a number of actions, including a reduction in crystal formation in the tubular fluid, inflammatory reaction control and interaction with oxidative stress. These factors lead concurrently to preserve tubular epithelial cell and renal interstitium integrity. In addition, these results suggest that the principal mechanism of action should be mediated by angiotensin II type 1 receptors.

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.

Cardiac mitochondrial function and tissue remodelling are improved by a non-antihypertensive dose of enalapril in spontaneously hypertensive rats.

Renal and cardiac benefits of renin-angiotensin system inhibition exceed blood pressure (BP) reduction and seem to involve mitochondrial function. It has been shown that RAS inhibition prevented mitochondrial dysfunction in spontaneously hypertensive rats (SHR) kidneys. Here, it is investigated whether a non-antihypertensive enalapril dose protects cardiac tissue and mitochondria function. Three-month-old SHR received water containing enalapril (10 mg/kg/day, SHR+Enal) or no additions (SHR-C) for 5 months. Wistar-Kyoto rats (WKY) were normotensive controls. At month 5, BP was similar in SHR+Enal and SHR-C. In SHR+Enal and WKY, heart weight and myocardial fibrosis were lower than in SHR-C. Matrix metalloprotease-2 activity was lower in SHR+Enal with respect to SHR-C and WKY. In SHR+Enal and WKY, NADH/cytochrome c oxidoreductase activity, eNOS protein and activity and mtNOS activity were higher and Mn-SOD activity was lower than in SHR-C. In summary, enalapril at a non-antihypertensive dose prevented cardiac hypertrophy and modifies parameters of cardiac mitochondrial dysfunction in SHR.

Sodium Intake Is Associated With Parasympathetic Tone and Metabolic Parameters in Mild Hypertension

BACKGROUND Although the impairment of parasympathetic cardiac control was described in hypertensives submitted to a high salt diet, the impact of this autonomic abnormality on metabolic and inflammation markers in patients with mild hypertension has not been explored. METHODS Four hundred and ninety mild essential hypertensive patients (144 ± 9/94 ± 9 mm Hg, 49.5 ± 13.9 years, 67.9 % male) were studied. Dietary sodium intake was estimated by measuring 24-h urinary sodium excretion (UNa), and the patients were classified according to UNa levels as follows: low (<50 mEq/l), medium (50-99 mEq/l), and high UNa (≥100 mEq/l). Parasympathetic tone was evaluated by assessing heart rate recovery (HRR) after an exercise stress test. HRR, plasma lipids, glucose metabolism, and inflammatory biomarkers were compared across UNa groups. RESULTS HRR and high-density lipoprotein (HDL)-cholesterol were progressively lower, and insulin (INS), homeostasis model assessment of insulin resistance (HOMAir), ultrasensitive-C-reactive protein (usCRP) were progressively higher across increasing UNa groups. In the low and medium UNa groups, HDL-cholesterol was higher and CRP was lower than that in high UNa (P < 0.01 and P < 0.05, respectively) (Dunnett post-hoc test). In the low UNa group, triglycerides (TGs), INS, and HOMAir were lower than that in high UNa (P < 0.05). Multiple linear regression analysis showed that UNa, HOMAir, and heart rate (HR) were negatively associated with HRR (P < 0.0001, P < 0.0001, and P = 0.001, respectively). CONCLUSIONS In the essential hypertensive patients studied high sodium intake is associated with parasympathetic inhibition, lipid disturbances, and inflammation. Studies designed to assess causality between sodium intake and metabolic and autonomic status are needed to evaluate the relevance of controlling sodium intake, especially in hypertensive patients.

Are plasma renin activity and aldosterone levels useful as a screening test to differentiate between unilateral and bilateral renal artery stenosis in hypertensive patients

Objective To evaluate the serum aldosterone (Ald)/plasmatic renin activity (PRA) ratio as a surrogate marker of renin–angiotensin–aldosterone system status in unilateral (Uni)- and bilateral (Bi)-renal artery stenosis (RAS). Methods Seven hundred and eight hypertensive patients (HTP) were studied. Intermediate and high pretest risk of RAS was detected in 66 HTP who subsequently underwent renal gadolinium-enhanced magnetic resonance and arteriography. After application of exclusion criteria 51 HTP remained: 16 with Uni-RAS, 16 with Bi-RAS and 19 essential hypertensives with normal arteries. Nineteen normotensive individuals were also studied. Ald and PRA were determined before and after stenosis resolution by balloon angioplasty and stent implantation. Results Ald/PRA (ng/dl per (ng/ml per h−1)) was markedly high in Bi-RAS (5.92 ± 2.30, P < 0.001), and markedly low in Uni-RAS (0.38 ± 0.17, P < 0.001) versus essential hypertensives (1.52 ± 2.02). Multilevel likelihood ratios for Bi-RAS were positive for Ald/PRA higher than 3.6, negative for Ald/PRA lower than 0.2, and neutral for Ald/PRA at least 0.2 and 3.6 or less. ROC analysis identified Ald/PRA lower than 0.5 and Ald/PRA higher than 3.7 to have the best sensitivity and specificity to detect Uni-RAS and Bi-RAS, respectively. In Uni-RAS, but not in Bi-RAS, postinterventional PRA was significantly lower than basal PRA. In Uni-RAS and Bi-RAS, postinterventional Ald was approximately 30% and approximately three times lower than basal Ald, respectively. In essential hypertensives, PRA and Ald showed no changes in the same period. Conclusion In the population studied, Ald, PRA and Ald/PRA were significantly different among essential hypertensives, and HTP with Uni-RAS or Bi-RAS. Studies with a higher number of patients will allow exploration of the usefulness of pharmacologic aldosterone blockade in Bi-RAS, and to assess the relevance of Ald/PRA to differentiate Uni-RAS from Bi-RAS.