Inés Stella

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.

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, Massons 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.

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.

The effects of angiotensin-converting-enzyme inhibitors on the fibrous envelope around mammary implants.

Background: Late capsular contraction around breast implants is one of the most difficult complications to prevent or resolve. The authors studied the mechanisms that control the fibrotic process in an animal model. Using angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonist, the authors previously described a significant reduction in fibrosis in different experimental models. Methods: Four groups of six rats each had a mini breast implant, 12 with a smooth surface and 12 with a textured surface. In two groups, the angiotensin-converting enzyme inhibitor enalapril was administered in drinking water, ad libitum, to determine its effect on both implant types. Two control groups were given plain drinking water. Three months postoperatively, all of the rats were killed and the capsule sections were cut and stained with hematoxylin and eosin and Masson’s trichrome. Immunolabeling of collagen III and transforming growth factor (TGF)-&bgr;1 was performed using monoclonal antibodies. Results: Significant differences were found between smooth and textured implants, with a uniformly low inflammatory response found on textured implants. For both surfaces, the enalapril-treated group had a significant reduction of the inflammatory process that was especially marked in the textured implants. Immunostaining for collagen III and TGF-&bgr;1 showed a consistent reduction in both fibrous tissue and cytokine mediator. Conclusions: Enalapril lowers the expression of fibrotic mediators, TGF-&bgr;1, inflammatory markers, anti-ED1, anti–collagen III monoclonals, and the periprosthetic fibrosis process. The reduction of TGF-&bgr;1 indicates that the probable main cytokine mediator of the fibrotic cascade is attenuated. This hypothesis may provide the basis for a safe and cheap therapeutic strategy with which to modify the capsular contracture that sometimes affects women with mammary implants.

Vascular Structure and Oxidative Stress in Salt-Loaded Spontaneously Hypertensive Rats: Effects of Losartan and Atenolol

BACKGROUND Renin-angiotensin system (RAS) modulation by high dietary sodium may contribute to salt-induced hypertension, oxidative stress, and target organ damage. We investigated whether angiotensin II (Ang-II) type 1 (AT1)-receptor blockade (losartan) could protect the aorta and renal arteries from combined hypertension- and high dietary salt-related oxidative stress. METHODS Spontaneously hypertensive rats (3-month-old, n = 10/group) received tap water (SHR), water containing 1.5% NaCl (SHR+S), 1.5% NaCl and 30 mg losartan/kg/day (SHR+S+L), or 50 mg atenolol/kg/day (SHR+S+A). Atenolol was used for comparison. Ten Wistar-Kyoto rats (WKY) were controls. Systolic blood pressure (SBP) was determined by tail plethysmography. After 5 months of treatment, vascular remodeling and oxidative stress (superoxide production and NAD(P)H-oxidase activity (chemiluminescence), malondialdehyde (MDA) content (high-performance liquid chromatography), endothelial nitric oxide synthase (eNOS) activity [(14)C-arginine to (14)C citrulline], CuZn-SOD activity (spectrophotometry)) were studied. RESULTS In SHR, salt-loading significantly aggravated hypertension, urinary protein excretion, intraparenchymal renal artery (IPRArt) perivascular fibrosis, aortic and renal artery oxidative stress, and induced endothelial cell loss in IPRArts. In salt-loaded SHR, 5-month losartan and atenolol treatments similarly reduced SBP, but only losartan significantly prevented (i) urinary protein excretion increase, (ii) or attenuated hypertension-related vascular remodeling, (iii) aortic MDA accumulation, (iv) renal artery eNOS activity lowering, and (v) aortic and renal artery superoxide dismutase (SOD) activity reduction. In SHR+S, the contributions to aortic superoxide production were as follows: uncoupled eNOS > xanthine oxidase (XO) > NAD(P)H oxidase. CONCLUSIONS In this salt-sensitive genetic hypertension model, losartan protects from hypertension- and high dietary salt-related vascular oxidative stress, exceeding the benefits of BP reduction. Also, during salt overload, BP-independent factors contribute to vascular remodeling, at least part of which derive from AT1-receptor activation.

Perinatal and infant early atherosclerotic coronary lesions.

OBJECTIVE Because the fetal origin of coronary artery lesions is controversial, early atherosclerotic coronary artery lesions in late fetal stillborns and infants, as well as the possible atherogenic role of maternal cigarette smoking, were studied. METHODS Twenty-two fetal death and 36 sudden infant death syndrome victims were examined by autopsy. In 28 of 58 cases, the mothers were smokers. Serially cut sections of coronary arteries were stained for light microscopy and immunotypified for CD68, CD34, alpha-smooth muscle actin, proliferating cell nuclear antigen, c-fos and apoptosis. RESULTS Multifocal coronary lesions were detected in 10 of 12 fetuses and in 15 of 16 infants whose mothers smoked. Arterial lesions in infants with nonsmoking mothers were observed in only five cases (two of 10 fetuses and three of 20 infants) (P<0.001). Alterations ranged from focal areas with mild myointimal thickening in prenatal life to early soft plaques in infants. Smooth muscle cells infiltrated into the subendothelium. These early lesions demonstrated c-fos gene activation in the smooth muscle cells of the media, and in some of these, positivity for apoptosis was observed, suggesting that c-fos overexpression may promote proliferation, as evidenced by proliferating cell nuclear antigen-positive cells. CONCLUSIONS Early intimal alterations of the coronary arteries are detectable in the prenatal and infancy period, and may be significantly associated with maternal smoking.

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.

Different Effect of Losartan and Amlodipine on Penile Structures in Male Spontaneously Hypertensive Rats

Background: Erectile dysfunction is highly prevalent in hypertensive patients. Since both angiotensin II receptor type-1 blockers (ARBs) and calcium antagonists are current and effective antihypertensive drugs, the aim of this study was to determine possible differences between ARBs and calcium antagonists concerning the protection of penile structures from the deleterious effects of arterial hypertension. Methods and Results: During 6 months, 3 groups of male spontaneously hypertensive rats (SHR) and 1 of Wistar-Kyoto (WKY) rats, as a control group, were studied: SHR without treatment; SHR with losartan (L) 30 mg/kg/day; SHR with amlodipine (A) 3 mg/kg/day, and WKY without treatment. Cavernous smooth muscle (CSM) and vascular smooth muscle (VSM) from cavernous arteries, cavernous tissue fibrosis and collagen type III (COL III) were evaluated. After 6 months, SHR+L and SHR+A showed a similar reduction in blood pressure compared with untreated SHR. However, only SHR+L and control WKY presented significantly lower values of: CSM (p < 0.01), VSM (p < 0.01), and COL III ( p < 0.01) when compared with either untreated SHR or SHR+A. There was also a positive correlation between left ventricular mass and proteinuria with VSM from cavernous arteries, CSM and COL III in untreated SHR and SHR+A. These relations were not present in SHR+L and WKY. Conclusion: Although losartan and amlodipine achieved similar blood pressure control, losartan but not amlodipine showed a significant protective role against structural changes in the vessels and cavernous spaces of the erectile tissue caused by arterial hypertension.

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.