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Enalapril and losartan attenuate mitochondrial dysfunction in aged rats¹

Physical Chemistry-PRALIB, School of Pharmacy and Biochemistry, University of Buenos Aires; and
^* Institute of Cardiovascular Research (ININCA), Buenos Aires, Argentina

²Correspondence: Junín 956, Buenos Aires (1113), Argentina. E-mail: cfraga{at}ffyb.uba.ar

SPECIFIC AIMS

Inhibition of the renin angiotensin system (RAS) can attenuate the effects of aging on renal structure and function. The aim of the present study was to investigate whether an angiotensin-converting enzyme inhibitor (enalapril) or an angiotensin II receptor blocker (losartan) could attenuate age-associated changes in kidney mitochondria.

PRINCIPAL FINDINGS

1. Enalapril and losartan attenuate age-related mitochondrial dysfunction
Respiratory control ratios were used as indicators of mitochondrial integrity and ADP/O ratios as an estimation of the capacity for energy production.

The present results confirm previous reports showing that mitochondria isolated from old untreated rats (22-month-old male Wistar rats) (Old) display an increased generation of hydrogen peroxide and a decline in the capacity for energy production compared with mitochondria isolated from young untreated rats (4-month-old) (Young) (Table 1 ). In mitochondria from 22-month-old rats that had received drinking water containing enalapril (10 mg· kg^-1·day^-1) (Enal) or losartan (30 mg· kg^-1·day^-1) (Los) for 8 months, production of hydrogen peroxide was lower than that observed in Old rats; the capacity to respond to a demand of energy was similar to that shown by Young rats (Table 1) . Mitochondrial NOS activity and uncoupling protein 2 (UCP2) level were lower (27 and 76%, respectively) in the Old relative to the Young group (Table 1) , but in Enal and in Los groups, mitochondrial NOS activity and UCP2 protein level were similar to that observed in the Young group. Mitochondrial Mn-SOD activity was significantly higher (70%) in the Old group than in any of the other groups (Table 1) .

2. Enalapril and losartan mitigate age-related mitochondrial ultrastructural changes in proximal tubular epithelial cells
Ultrastructural analysis of the kidney focused on proximal tubular epithelial cells because they have a large number of mitochondria, and are highly dependent on mitochondrial energy production for proper function. In proximal tubules from the Young group, the totality of epithelial cells examined showed good preservation of microvilli. In the Old group, the number of epithelial cells showing an absence of microvilli (Table 2 ) or altered microvilli was higher than in the Enal or Los groups. In the Old group, the number of mitochondria per cell was lower than in the Enal, Los, and Young groups. The number of cristae per unit length of mitochondrial contour was similar in the groups studied. However, in the Old group the number of mitochondria showing poorly defined cristae was higher than in the Enal, Los, and Young groups. In the Old group, 90% of tubular epithelial cells showed osmiophilic bodies enclosed within the basal infoldings of proximal tubular epithelial cells, as opposed to 40% (P<0.05) in the Enal and Los groups. In the Young group, osmiophilic bodies were virtually absent from tubular epithelial cells. In the Old group the number of osmiophilic bodies per cell was higher than in the Enal, Los, and Young groups.

3. Enalapril and losartan treatments attenuate age-related oxidation of kidney glutathione
GSH+ GSSG content in the kidney was similar in the Old, Enal, and Young groups and higher in the Los group than other groups (54, 47, and 47% vs. Old, Enal, and in the Young, respectively). In the Old group, kidney GSH/GSSG was lower (80%) than in the Young group. In the Enal and Los groups, GSH/GSSG was higher (132 and 210%, respectively) than in the Old group.

CONCLUSIONS

The present results show that long-term inhibition of the RAS can attenuate several age-associated alterations in mitochondrial function and structure. Enalapril and losartan prevented the age-associated increase in systolic blood pressure. Both treatments prevented age-associated decline of kidney function, as evaluated by plasma creatinine and urinary albumin excretion. Enalapril and losartan treatments preserved kidney mitochondrial function from the effects of aging. This protection was revealed by the capacity of both drugs to prevent an age-associated decline in the capacity for energy production and to attenuate the age-associated increase in mitochondrial oxidant production. Mitochondrial respiration is a major subcellular source of O₂^•- and H₂O₂, two species that promote oxidative damage to macromolecules. The mitochondrial theory of aging proposes that oxidant-induced mitochondrial degeneration leads to a decrease in mitochondria’s capacity to respond to an energy demand, conducive to a progressive decline of cell function. Enalapril and losartan could protect mitochondria from the effects of aging by attenuating and/or reversing oxidant-induced damage to mitochondrial components, as suggested by the observed lower production of H₂O₂ and the maintenance of an adequate ADP/O ratio in old rats treated with the RAS inhibitors.

Nitric oxide (NO) has been proposed to modulate mitochondrial function. Enalapril and losartan can increase endothelial NO generation. In this study, we show that both the enalapril and losartan treatments prevented the decrease in NOS activity observed in mitochondria from Old rats. Current evidence points to the concept of UCP2 acting as an oxidant stress-compensating mechanism, and a role for UCP2 in cellular pathophysiological processes that involve ROS has been suggested. Several agents that up-regulate NOS also increase UCP expression, possibly to prevent excessive O₂^•- production. Modulation of mtNOS activity and UCP2 protein level suggests that the protective action that enalapril and losartan exert on mitochondrial function and structure may rely on a mechanism that involves both NO and UCP2. NO can mediate cell-protective or cell-damaging reactions depending on the relative levels of O₂, NO, O₂^•-, H₂O₂, and other oxidants. Based on the observed preservation of kidney mitochondrial structure and function, it can be assumed that in enalapril- or losartan-treated animals the higher levels of NO, as well as the lower levels of H₂O₂ and O₂^•- compared with untreated Old animals, led to metabolization of NO through nondamaging routes. The present data may be interpreted by considering the role that mitochondria have as active participants in cell signal transduction pathways, particularly with regard to the modulatory role of NO on H₂O₂ production and O₂ metabolism. The modulation of mitochondrial respiratory rates and mitochondrial signaling by NO may affect cell proliferation, growth, and apoptosis, thereby contributing to the maintenance of tissue structure and function. In Old untreated animals, loss of control of NO over respiration and H₂O₂ production may have led to structural and functional changes in the kidney. The main findings and proposed routes conducting to the observed enalapril-or losartan-mediated mitochondrial effects are summarized in Fig. 1 .

View larger version (30K): [in this window] [in a new window]   Figure 1. Routes proposed to explain the protective effects of enalapril or losartan treatments against age-related decline of mitochondrial function and structure showing effects observed in Old rats (Aging) and old rats treated with enalapril or losartan (Aging+RASi).

It has been reported that NO can increase mitochondrial H₂O₂ generation. This discrepancy with the present data, showing an increase in mtNOS activity in concurrence with a decrease in H₂O₂ production, may stem from the fact that in the studies cited above, stimulators of NOS were added to isolated mitochondria, a system where regulation of UCP2 expression is not possible. Consequently, the in vitro inhibition of respiration by NO brings about an increase of ROS (H₂O₂) production that cannot be compensated for by UCP2 up-regulation and consequent attenuation of O₂^•- and H₂O₂ formation. In the current study, the in vivo enalapril and losartan treatments increase mtNOS activity in a setting where mitochondrial/nuclear cross-talk is possible and, by eliciting UCP2 up-regulation, can lower H₂O₂ production, possibly leading to a better preservation of mitochondrial function and structure.

In mitochondria, Mn-SOD activity showed an age-related increase that was attenuated by enalapril and losartan treatments, suggesting that in the Old group Mn-SOD activity may have been up-regulated to counteract an augmentation in O₂^•- generation, as indicated by the increase in H₂O₂ production.

Ultrastructural analysis of kidney tubular epithelial cells showed that enalapril and losartan treatments can both mitigate the age-related decrease in number of mitochondria as well as alterations in mitochondrial structure and distribution observed in Old rats. Enalapril and losartan treatments also attenuate the age-associated accumulation of osmiophilic bodies in proximal tubular epithelial cells of the kidney. Enhanced osmiophilia probably indicates an increase in the storage of lipids, an early event in mitochondrial degeneration. In addition, enalapril and losartan treatments prevented the age-related effacement of proximal tubular cell microvilli. The maintenance of the architecture of a cell depends at least in part on an adequate provision of energy. In consequence, the preservation of microvilli by enalapril and losartan treatments might be related to the ability of these drugs to prevent the age-associated decline in the capacity of mitochondria to generate ATP.

The role of glutathione as an antioxidant depends not only on the glutathione pool size but on its reduction/oxidation status. Kidney GSH/GSSG ratios showed that in the Old group, glutathione was relatively more oxidized than in the Young group, and enalapril and losartan treatments both attenuated the age-related oxidation of kidney glutathione. Losartan enhanced total glutathione content. The administration of antioxidants has been shown to protect against several age-associated mitochondrial changes including oxidant damage to mitochondrial DNA, GSH oxidation, increased peroxide generation, and impairment of both mitochondrial function and morphology. Taken together, these results suggest that exogenously administered antioxidants can make their way to mitochondria or improve the tissues/cells antioxidant status, protecting these organelles from oxidant damage. In the same way that exogenous antioxidants can protect mitochondria from the effects of aging, enalapril- and losartan-mediated enhancement of glutathione content and redox status could protect these organelles from oxidants upon aging.

Current findings showing that enalapril or losartan can protect against both age-related mitochondrial dysfunction (decreased capacity of energy production and increased H₂O₂ generation) and mitochondrial ultrastructural changes in the kidney afford an alternative mechanism to explain the health-related effects of RAS inhibitors. That these two compounds that act at different levels in the RAS produce a similar pattern of changes is indicative of a physiological relationship between the RAS and age-associated cell deterioration. Further experiments will define whether the association seen between RAS and mitochondria is related to or occurs independent of the modulation of the blood pressure.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0063fje; to cite this article, use FASEB J. (April 22, 2003) 10.1096/fj.02-0063fje

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