Angiotensin-(1–7) stimulates oxidative stress in rat kidney

Regulatory Peptides 106 (2002) 67 – 70 www.elsevier.com/locate/regpep

Angiotensin-(1–7) stimulates oxidative stress in rat kidney Soledad Gonzales a, Guillermo O. Noriega a, Marı´a L. Tomaro a,*, Clara Pen˜a a,b a

Departamento de Quı´mica Biolo´gica, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Junı´n 956, Buenos Aires 1113, Argentina b Instituto de Quı´mica y Fisicoquı´mica Biolo´gicas, Facultad de Farmacia y Bioquı´mica, Universidad de Buenos Aires, Buenos Aires, Argentina Received 17 September 2001; received in revised form 4 February 2002; accepted 8 February 2002

Abstract The effect of two different doses of angiotensin-(1 – 7) and angiotensin II on the oxidative stress generation was analyzed in rat kidney. Animals were injected intraperitoneally with a single dose of angiotensin-(1 – 7) or angiotensin II (20 or 50 nmol/kg body weight) and killed 3 h after injection. Production of thiobarbituric acid reactive substances (TBARS), measured as indicator of oxidative stress induction, was significantly increased in rat kidney after Ang-(1 – 7) administration up to 30% and 50% over controls, at 20 and 50 nmol/kg, respectively. Reduced glutathione (GSH), the most important soluble antioxidant defense in mammalian cells, showed a significant decrease of 13% and 20% at 20 and 50 nmol/kg of angiotensin-(1 – 7), respectively. When the antioxidant enzyme activities were determined, it was found that catalase activity was not altered by the assayed angiotensin-(1 – 7) doses while superoxide dismutase and glutathione peroxidase activities were significantly reduced by injection of 20 nmol/kg (34% and 13%, with respect to controls) and 50 nmol/kg of angiotensin-(1 – 7) (54% and 22%, respectively). In contrast, angiotensin II injections did not produce significant changes neither in TBARS levels nor in soluble and enzymatic defense parameters at the two doses used in this work. These results suggest that angiotensin-(1 – 7) is undoubtedly related to oxidative stress induction. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Angiotensin peptides; Oxidative damage; Reduced glutathione; Enzymatic antioxidant defenses

1. Introduction The effector hormone angiotensin (Ang) II was considered over the years as the main component of the renin – angiotensin system. Nowadays, it is well established that other members of this peptide family have unique biological effects [1]. The heptapeptide Ang-(1 –7) produced by carboxyl-terminal degradations of Ang I or Ang II has been detected in different tissues from rats, dogs and human beings [2 –5] and elicits effects that may mimic or oppose those produced by Ang II [6]. Ang-(1– 7), like Ang II, stimulates vasopressin release in rat hypothalamus [7] and facilitates peripheral noradrenergic neurotransmission [8]. Unlike Ang II, the heptapeptide is not a vasopressor and does not stimulate dipsogenesis or aldosterone release [1,6]. Moreover, it inhibits Ang-converting enzyme [9], which cleaves Ang II from Ang I, and activates antihypertensive mechanisms. One of the most important actions of Ang-(1– 7) identified so far is related to the control of hydroelectrolyte balance [10] modu-

lating urinary water and sodium excretion [11]. Immunohistochemistry demonstrated that the peptide is present in brain areas related to the control of hydromineral balance [2]. At least one site of action is at the level of the proximal tubule, where Ang-(1 –7) has a potential role in the regulation of sodium transport [12]. It has been described a modulatory role of Ang-(1– 7) for renal Na+, K+-ATPase [13] and a reduction in energy-dependent transcellular sodium transport [12]. The present investigation was undertaken to determine if the complex renal activity displayed by Ang-(1– 7) is accompanied in rat kidney by oxidative stress stimulation and to compare its effects with those shown by Ang II. Lipid peroxidation measured as TBARS content was analyzed. In addition, the profile of water-soluble antioxidant, reduced glutathione, and the activity of antioxidant enzymes were also determined.

2. Materials and methods 2.1. Chemicals

*

Corresponding author. Tel.: +54-11-4964-8237; fax: +54-11-5083645. E-mail address: [email protected] (M.L. Tomaro).

NADPH, reduced glutathione (GSH), oxidized glutathione (GSSG), 5,5V-dithio-bis-(2-nitrobenzoic acid) (DTNB),

0167-0115/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 0 11 5 ( 0 2 ) 0 0 0 3 2 - 0

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S. Gonzales et al. / Regulatory Peptides 106 (2002) 67–70

angiotensin-(1 –7), angiotensin II, thiobarbituric acid, glutathione reductase and 2-vinylpyridine were from Sigma (St. Louis, MO); ter-butyl hydroperoxide was from Aldrich Chemical (Phillipsburg, NJ). All other chemicals were of analytical grade. 2.2. Animals and treatments Female albino Wistar rats (180 – 200 g) were injected intraperitoneally with a single dose of Ang-(1 – 7) or Ang II (20 or 50 nmol/kg body weight) dissolved in saline solution. Controls were carried out by injection of saline solution. 2.3. Enzyme preparations and assays Rats were killed 3 h after Ang-(1– 7) or Ang II injection. Kidneys were excised, washed with an ice-cold saline solution (0.9% NaCl) and homogenized in a Potter-Elvehejm homogenizer. Superoxide dismutase, catalase and glutathione peroxidase activities were determined spectrophotometrically in kidney homogenates prepared in a medium consisting of 140 mM KCl and 25 mM potassium phosphate buffer (pH 7.4), and centrifuged at 600g for 10 min. The supernatant, a suspension of preserved organelles, was used as homogenate. Catalase activity was determined by measuring the decrease in absorbance at 240 nm [14], glutathione peroxidase activity following NADPH oxidation at 340 nm [15], and superoxide dismutase (SOD) activity by inhibition of adrenochrome formation rate at 480 nm [16]. One unit in the SOD assay is defined as the amount of enzymatic protein required to inhibit 50% of epinephrine auto-oxidation. 2.4. Lipid peroxidation Homogenate lipid peroxidation was determined by measuring the rate of production of thiobarbituric acid reactive substances (TBARS) (expressed as malondialdehyde equivalents). One volume of homogenate was mixed with 0.5 volume trichloroacetic acid (15% w/v) and centrifuged at

Fig. 2. Effect of angiotensin-(1 – 7) or angiotensin II on GSH content. Rats were treated as described in Materials and Methods and killed at the indicated time. Values are the mean from nine rats and bars indicate S.E.M. *Significant differences ( P < 0.05) according to Student’s t-test.

2000g for 10 min. Supernatant (1 ml) was mixed with 0.5 ml thiobarbituric acid (0.7% w/v) and boiled for 10 min. After cooling, sample absorbance was determined spectrophotometrically at 535 nm. Malondialdehyde concentration was calculated using an e value of 1.56105 M 1 cm 1 [17]. 2.5. Endogenous hepatic GSH content Total glutathione (GSH plus GSSG) was determined in kidney homogenates after precipitation with 2% perchloric acid and using yeast-glutathione reductase, DTNB and NADPH, at 340 nm. GSSG was determined by the same method in the presence of 2-vinylpyridine and GSH calculated as the difference between total glutathione and GSSG [18]. 2.6. Protein determination Protein concentration was evaluated by the method of Lowry et al. [19] using bovine serum albumin as standard.

Table 1 Effect of angiotensin-(1 – 7) on antioxidant enzymes activities Treatments Control

Angiotensin-(1 – 7) Angiotensin-(1 – 7) (20 nmol/kg) (50 nmol/kg)

Superoxide dismutase 4.1F0.4 2.7F0.3* (U/mg protein) Catalase 18.2F1.0 20.1F2.0 (pmol/mg protein) Glutathione peroxidase 0.24F0.01 0.21F0.01* (U/mg protein)a

Fig. 1. Effect of angiotensin-(1 – 7) or angiotensin II on lipid peroxidation. Rats were treated as described in Materials and Methods and killed at the indicated time. Values are the mean from nine rats and bars indicate S.E.M. *Significant differences ( P < 0.05) according to Student’s t-test.

1.9F0.2* 21.3F2.1 0.19F0.01*

Control animals were injected with a single dose of saline solution. Angiotensin-(1 – 7) was administered intraperitoneally at the indicated doses. Rats were killed 3 h after the start of the experiment and the enzymatic activity was assayed as described in the text. a One unit of the enzyme represents the decrease of 1 mmol of NADPH/ min under assay conditions. Data are means F S.E.M. of three different experiments using three rats each time. * Significant differences ( P < 0.05) as assessed by Student’s t-test.

S. Gonzales et al. / Regulatory Peptides 106 (2002) 67–70

2.7. Statistics Figures in the text and tables indicate mean values F S.E.M. Differences between control and treated animals were analyzed using Student’s t-test, taking P<0.05 as significant.

3. Results and discussion Oxidative stress is mediated by reactive oxygen species (ROS), including superoxide anion (O2 ), hydrogen peroxide (H2O2), and hydroxyl radical (HO) which occur in tissues and may damage DNA, proteins, carbohydrates, and lipids. These potentially deleterious reactions are controlled by a system of antioxidant defenses, which eliminate pro-oxidants and scavenge free radicals. Protection against oxidation is provided by various intracellular compounds such as glutathione, and antioxidant enzymes including catalase, superoxide dismutase, and glutathione peroxidase [20]. However, oxygen toxicity emerges when the production of ROS exceeds the quenching capacity of natural cellular protective systems. Several physical and chemical agents are involved in oxidative stress induction in vivo, but up to now this behavior has not yet been described for Ang-(1 –7). Even when lipid peroxides are the major contributors to TBARS generation, it has been shown that malondialdehyde (and its analogous) is not only formed during peroxidation of lipids. Oxidative stress induces the degradation of a variety of important biological molecules such as amino acids, proteins and carbohydrates, with the consequent release of malondialdehyde [21]. Therefore, the increase in TBARS content is more precisely an indicator of a general Ang-(1– 7)-induced oxidative damage due to the impairment of cell defense system. When the Ang-(1 –7)-induced oxidative stress was analyzed, significant differences were observed in rat kidney subjected to Ang-(1– 7) treatments (30% and 50% over controls, at 20 and 50 nmol/kg body weight, respectively) (Fig. 1). Glutathione is the most important cellular thiol acting as a substrate for several transferases, peroxidases and other enzymes that prevent or mitigate the deleterious effects of oxygen free radicals [22]. After Ang-(1– 7) administration, a significant decrease in GSH content at the two used doses was observed: 13% and 20%, with respect to the control values (Fig. 2). Superoxide dismutases (SOD) are a group of enzymes that accelerate the conversion of O2 to H2O2 [20]. As shown in Table 1, an impressive decrease in renal superoxide dismutase activity was found under Ang-(1 – 7) injections of 20 or 50 nmol/kg body weight (34% and 54%, with respect to controls). Catalase, glutathione peroxidase, and a variety of general peroxidases catalyze the breakdown of H2O2 [20]. Compared to controls, glutathione peroxidase activity decreased 13% and 22% in response to Ang-(1– 7) injections at 20 or

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Table 2 Effect of angiotensin II on antioxidant enzymes activities Treatments Control Superoxide dismutase (U/mg protein) Catalase (pmol/mg protein) Glutathione peroxidase (U/mg protein)a

Angiotensin II (20 nmol/kg)

Angiotensin II (50 nmol/kg)

5.5F0.5

6.0F0.5

5.8F0.4

19.1F0.9

19.3F0.7

19.7F0.8

0.22F0.02

0.22F0.01

0.21F0.02

Control animals were injected with a single dose of saline solution. Angiotensin II was administered intraperitoneally at the indicated doses. Rats were killed 3 h after the start of the experiment and the enzymatic activity was assayed as described in the text. a One unit of the enzyme represents the decrease of 1 mmol of NADPH/ min under assay conditions. Data are means F S.E.M. of three different experiments using three rats each time.

50 nmol/kg body weight, respectively (Table 1), whereas catalase activity remained unchanged with respect to control values by both Ang-(1 – 7) treatments (Table 1). These findings are in line with the reported superoxide and nitric oxide generation by Ang-(1 –7) in cultured bovine aortic endothelial cells [23]. Recent work has shown that reactive oxygen species are involved in transducing many Ang II effects [24] and several authors have demonstrated oxidative effects for Ang II in cultured renal cells [25,26]. Likewise, Haugen et al. [26] reported that chronic administration of Ang II imposes oxidative stress on the kidney in vivo. Contrasting with these findings, under our experimental conditions, injection of Ang II did not produce significant changes neither in TBARS levels nor in soluble and enzymatic defense parameters at the two acute doses used in this study (Table 2). It is likely that the different results are due to differences between acute and chronic effects of Ang II. In summary, our results clearly demonstrate that acute administration of Ang-(1 – 7), but not Ang II, produces oxidative damage in rat kidney. Nevertheless, time course studies will be necessary to assess the biochemical mechanisms by which Ang-(1– 7) acts as oxidative stress inductor and the physiological significance of this action.

Acknowledgements This work was supported by grants from Universidad de Buenos Aires (Argentina) and Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas (CONICET) (Argentina). C.P. and M.L.T. are career investigators from CONICET.

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