Impact of angiotensin-converting enzyme inhibition on renal cortical nitrotyrosine content during increased extracellular glucose concentration

Clinical Biochemistry 39 (2006) 633 – 639

Impact of angiotensin-converting enzyme inhibition on renal cortical nitrotyrosine content during increased extracellular glucose concentration Naohito Ishii a , Hideki Ikenaga b , Pamela K. Carmines c , Nobukazu Takada a , Toshio Okazaki d , Tatsuo Nagai d , Tadakazu Maeda e , Yoshikazu Aoki f , Takao Saruta g , Masato Katagiri a,⁎ a

Department of Clinical Physiology, Kitasato University School of Allied Health Sciences, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan b Ikenaga Clinic, Tochigi, Japan c Department of Cellular and Integrative Physiology, University of Nebraska College of Medicine, Omaha, NE 68583, USA d Department of Hematological Informatics, Kitasato University School of Allied Health Sciences, Kanagawa, Japan e Department of Physics, Kitasato University School of Science, Kanagawa, Japan f Kanagawa Health Service Association, Kanagawa, Japan g Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan Received 11 September 2005; received in revised form 31 January 2006; accepted 21 February 2006 Available online 21 April 2006

Abstract Objectives: Experiments evaluated the hypothesis that angiotensin-converting enzyme (ACE) inhibition suppresses hyperglycemia-induced nitrotyrosine (NT) production in the renal cortex. Design and methods: Rats were untreated (UNTR, n = 6) or received the ACE inhibitor enalapril (20 mg/kg/day; ENAL, n = 6) for 2 weeks. Renal cortical slices were incubated for 90 min in media containing 5 (normal) or 20 mmol/L (high) glucose. Superoxide anion (O2·−) and nitrate + nitrite (NOX) levels were measured in the media. Superoxide dismutase (SOD) activity and NT content were measured in the tissue homogenate. Results: In the UNTR group, high glucose increased O·2− and NOX production by the renal cortex (P < 0.05 vs. normal glucose). Likewise, NT content and SOD activity of the renal cortex augmented (P < 0.05 vs. normal glucose). In the ENAL group, O2·− production and NT content were glucose-insensitive, but high glucose exerted an exaggerated impact on NOX production and SOD activity (P < 0.01 vs. UNTR in high glucose). Conclusion: Accelerated NT content in the renal cortex during high-glucose conditions was prevented by ACE inhibitor treatment. It was suggested that, apart from its anti-hypertensive effect, the mechanism of suppressed NT degradation in the renal cortex by the ACE inhibitor enhances both O2·− degradation per se and antioxidative effects including SOD activation. © 2006 The Canadian Society of Clinical Chemists. All rights reserved. Keywords: Diabetes mellitus; Angiotensin-converting enzyme inhibition; Superoxide anion; Nitric oxide; Peroxynitrite; Superoxide dismutase; Protein tyrosine nitration

Introduction Diabetes mellitus (DM) is well known to provoke serious complications, including diabetic nephropathy as one of the major focuses in current research of kidney diseases [1]. In diabetic nephropathy patients, glomerular filtration rate (GFR) and microalbuminuria are increased in the early stages. Then, GFR reduction and proteinuria elevation with deterioration of ⁎ Corresponding author. Fax: +81 42 778 9628. E-mail address: [email protected] (M. Katagiri).

renal function gradually develop into chronic renal failure. This leads to chronic renal failure that requires either hemodialysis or renal transplantation for survival. Nevertheless, the pathophysiological mechanisms underlying development of diabetic nephropathy remain unexplained. The superoxide anion (O2·−), a major free radical, is formed by various reactions such as those involving NAD(P)H oxidase, xanthine oxidase or the mitochondrial electron transport chain. O2·− causes inflammation, cell growth, apoptosis and damage to major cell constituents during conditions of oxidative stress [2–5]. Nitric oxide (NO) is also considered to represent a free radical that plays versatile

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physiological and pathophysiological roles through its actions as a vasodilator, neurotransmitter and bactericide [6–9]. During the early stage of DM, accelerated renal cortical O2·− production promotes NO degradation via formation of the powerful toxic oxidant peroxynitrite (O2·− + NO → ONOO−), resulting in nitration of protein tyrosine residues (nitrotyrosine, NT) [10,11]. Thus, ONOO− and NT production may contribute to the development of diabetic nephropathy [12,13]. Angiotensin-converting enzyme (ACE) inhibitors are now recommended as an initial therapy for DM because of their ability to decelerate development of diabetic nephropathy with or without hypertension [12,14–18]. An antioxidant effect of ACE inhibitors may contribute to the ability of these agents to prevent diabetic nephropathy. Indeed, enalapril (an ACE inhibitor) has been reported to increase antioxidant enzyme activity in the kidneys of rats with DM [19,20]. Accordingly, we hypothesized that ACE inhibition would suppress NT production in the renal cortex under hyperglycemic conditions. The validity of this postulate was explored (by measurement of NO and O2·− production, NT content and superoxide dismutase (SOD) activity) in renal cortical slices incubated in two different glucose conditions, normal (5 mmol/L, equivalent to the blood glucose concentration of normal rats) and high-glucose (20 mmol/L, equivalent to the blood glucose concentration of streptozotocine-induced DM rats) [10], using tissue harvested from untreated and enalapril-treated rats.

Total NOX production assay Renal cortical slices were incubated at 37°C for 90 min in Hank's balanced salt solution (HBSS) containing 500 U/mL SOD (Sigma Chemical Co. to minimize NO consumption by reaction with O2·−) and either 5 or 20 mmol/L glucose. The supernatant was removed at 90 min and stored at −80°C until NOX production measurement. Slices from each kidney were also incubated in media containing 10 mmol/L Nω-nitro-Larginine (NNA; Sigma Chemical Co.). The renal cortex was weighed, minced and homogenized (Ultra-Turrax T8; IKA Works, Inc., Staufen, Germany) in ice-cold HBSS. After 20 min of treatment of the homogenate in 10 mmol/L 3-[(3-cholamidopropyl) dimethlammonio] propanesulfonic acid (CHAPS, Sigma Chemical Co.) at 4°C and subsequent centrifugation at 10,000 × g, samples were stored at −80°C until measurement of protein concentrations. After storage, the supernatant was centrifuged at 10,000 × g at 4°C. NOX concentration in the centrifuged renal cortical supernatant was measured using the Griess assay (nitrite + nitrate assay kit-C II, Dojindo Laboratories, Kumamoto, Japan) [21]. NNA-sensitive nitrite + nitrate (NOX) production is presented as nmol/mg protein/90 min. As the Griess reagent also reacts with NNA, we made a correction to control for sample NNA concentration. O2·− production assay

Materials and methods Animals The Kitasato University Institutional Animal Care and Use Committee approved all procedures utilized in this study. Male Sprague–Dawley rats (Japan Clea, Inc., Tokyo, Japan) were randomly assigned into two groups—untreated rats (UNTR group) and rats receiving 20 mg/kg/day enalapril maleate (Sigma Chemical Co., St. Louis, MO, USA) for 2 weeks via their drinking water (ENAL group). The blood glucose concentration (GLUTEST ACE GT-1640; Sanwa Kagaku Kenkyusho Co., Ltd., Nagoya, Japan) and body weight were measured at 2- to 3day intervals. Systolic blood pressure was evaluated by tail plethysmography (model MK-2000; Muromachi Kikai Co., Ltd., Tokyo, Japan). Animals were housed individually in metabolic cages for the 2 days preceding the terminal experiment. The volume of urine collected during the final 24 h was determined gravimetrically. Urine samples were centrifuged and stored at −80°C until measurement of creatinine concentrations. In each group, rats were anesthetized with pentobarbital sodium (50 mg/kg i.p.; Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan). The abdominal aorta was cannulated, allowing the kidneys to be flushed with 20 U/mL heparinized saline. Each kidney was excised and weighed. Two medial slices were obtained from each kidney with a Stadie–Riggs microtome. The medullary portion of each slice was carefully removed and discarded. The cortical slices thus obtained were used for measurement of nitrite + nitrate (NOX) and O2·− production, SOD activity and NT content.

Renal cortical slices were incubated at 37°C for 90 min in HBSS containing 80 μmol/L cytochrome c, 10 mmol/L NNA (to minimize O2·− consumption via reaction with NO) and either 5 or 20 mmol/L glucose. To control for nonspecific reduction of ferricytochrome c, each of these treatments was imposed in both the absence and presence of 500 U/mL SOD. As a positive control, slices from each kidney were also incubated in the presence of 250 μg/mL heat-aggregated bovine IgG, which stimulates O2·− production by mesangial and proximal tubular cells [22]. The supernatant was removed at 90 min for measurement of O2·− production. The renal cortex was weighed, minced, homogenized (as described above) and stored at −80°C until measurement of protein concentrations. O2·− production was measured based on its ability to reduce ferricytochrome c, according to the established method [10,20]. The supernatant was removed at 90 min after initiating the cytochrome c incubation, centrifuged (10,000 × g) to remove any cellular debris, and absorbance was measured at 550 nm. The extinction coefficient of cytochrome c was assumed to be 2.1 × 104 L mol−1 cm−1. SOD-sensitive O2·− production was expressed as nanomoles reduced cytochrome c per mg protein per 90 min. SOD activity and NT assays Renal cortical slices were incubated at 37°C for 90 min in HBSS containing either 5 or 20 mmol/L glucose. After incubation, the renal cortical slices were utilized for SOD activity, NT and protein assays.

N. Ishii et al. / Clinical Biochemistry 39 (2006) 633–639 Table 1 Characteristics of normal (UNTR) and enalapril-treated (ENAL) rats

Body weight (g) Kidney weight (g) Right Left Blood glucose (mmol/L) Systolic blood pressure (mm Hg) Water intake (mL/day) Urine flow (mL/day) Creatinine excretion (μmol/day)

UNTR (n = 6)

ENAL (n = 6)

271.6 ± 3.6

259.0 ± 1.7

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performed utilizing the StatView software (SAS Institute, Cary, NC, USA) for a Macintosh computer. P values <0.05 were considered significant. All data are reported as means ± SEM. Results

1.25 ± 0.03 1.21 ± 0.09 5.8 ± 0.2 128 ± 9 31.3 ± 1.4 10.0 ± 0.9 120 ± 9

1.20 ± 0.04 1.17 ± 0.03 6.0 ± 0.3 126 ± 3 28.0 ± 1.8 8.9 ± 0.8 111 ± 9

Results are means ± SEM.

After storage, the renal cortex was weighed, minced and homogenized in ice-cold HBSS with 10 mmol/L CHAPS. The homogenate was centrifuged (10,000 × g), and the supernatant was stored at −80°C until the assay for SOD activity, according to the modified method of Bulucu et al. [23]. Briefly, the sample was incubated in a buffer solution (pH 10.2) containing 0.04 mmol/L xanthine sodium, 0.02 mmol/L 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT), 0.8 mmol/L EDTA, 42.5 mmol/L N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), and the 10 U/L xanthine-oxidaseinduced increase in absorbance at 505 nm was recorded. One unit of activity is defined as the amount of SOD required to inhibit the INT reduction rate of xanthine oxidase by 50% at 37°C. Free NT content in the renal cortex was determined using HPLC with electrochemical detection, modified from the method of Imam et al. [24]. Briefly, the renal cortex was weighed, minced, homogenized and sonicated in ice-cold 10 mmol/L sodium acetate buffer (pH 6.5) with 10 mmol/L CHAPS. The homogenates were centrifuged (10,000 × g), and supernatants were treated with 5 mg/mL pronase (protease type XIV, EC 3.4.24.31, Sigma Chemical Co.) for 20 h at 50°C. Enzymatic digests were treated with 20% trichloroacetic acid, centrifuged at 14,000 × g for 10 min at 4°C and passed through a 0.2 μm polyvinylidene difluoride filter before injection onto HPLC with an electrochemical detector (applied 1500 mV, EC-8020; TOHSO Corporation, Tokyo, Japan). The analytical column was TSK-GEL ODS80Ts (4.6 mm × 25 cm, TOHSO Corporation). The mobile phase was 50 mmol/L acetate/50 mmol/L citrate/5% (v/v) methanol, pH 3.1. Free NT concentration of renal cortex was expressed as pmol/mg protein.

Animal characteristics Table 1 summarizes physiological characteristics of the animals used in the present study, measured at the 2-week time point in the UNTR and ENAL groups. There were no significant differences in any of these measured parameters, indicating that the responses evident in the in vitro studies cannot be attributed to chronic enalapril-induced alterations in arterial pressure or other basic systemic parameters. NOX production Fig. 1 shows the effect of enalapril on NOX production by renal cortical slices in media containing 5 or 20 mmol/L glucose. NOX production was similar in tissue from UNTR and ENAL groups during incubation in the presence of 5 mmol/L glucose. Incubation of renal cortical tissue from UNTR rats in media containing 20 mmol/L glucose yielded NOX production values approximately 2 times those evident during incubation in 5 mmol/L glucose (P < 0.05). NOX production in the presence of 20 mmol/L glucose was 50% higher in the ENAL group than in the UNTR group (P < 0.05). Moreover, within the ENAL group, renal cortical NOX production during incubation in high-glucose media was 3 times that observed during incubation in 5 mmol/L glucose. Thus, enalapril pretreatment exaggerated the high-glucoseinduced renal cortical nitrosative stress.

Protein and creatinine assays Protein concentration in homogenized renal cortex was determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories; Hercules, CA, USA). Creatinine concentration in urine samples was measured by the Jaffe method [25]. Statistics Data were analyzed by ANOVA for repeated measures or unpaired t tests, as appropriate. All statistical computations were

Fig. 1. Effect of extracellular glucose concentration on NOX production by renal cortical slices from normal (UNTR) and enalapril-pretreated (ENAL) rats. Renal cortical slices were incubated at 37°C for 90 min in media containing 500 U/mL SOD, either 5 mmol/L or 20 mmol/L glucose. Values are means ± SEM. *P < 0.05 or **P < 0.01 vs. 5 mmol/L glucose within the UNTR or ENAL groups, †P < 0.05 vs. UNTR at the same glucose concentration.

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O2· − production Fig. 2 shows the effect of enalapril on O2·− production by renal cortical slices in media containing 5 or 20 mmol/L glucose. In tissue from UNTR rats, inclusion of aggregated IgG in the 5 or 20 mmol/L glucose media yielded O2·− production values averaging 3.2 ± 0.7 and 2.9 ± 0.4 nmol/mg protein/90 min, respectively (positive control). O2·− production by renal cortical slices from UNTR rats incubated in media containing 20 mmol/L glucose averaged approximately 2 times greater than values observed in 5 mmol/L glucose (P < 0.05). In cortical slices from ENAL rats, O2·− production in 5 mmol/L glucose media was similar to the UNTR group; however, the accelerated O2·− production observed in UNTR tissue under high-glucose conditions was prevented by enalapril pretreatment (P < 0.01 vs. the UNTR group in 20 mmol/L glucose; no significant difference from the ENAL group in 5 mmol/L glucose). SOD activity Fig. 3 illustrates the effect of chronic enalapril treatment on SOD activity of renal cortical slices in media containing 5 or 20 mmol/L glucose. SOD activity of renal cortex from the UNTR group in media containing 20 mmol/L glucose averaged 70% greater than that evident after 5 mmol/L glucose incubation (P < 0.05). SOD activity measured after incubation in either 5 or 20 mmol/L glucose conditions was substantially elevated in the ENAL group, averaging nearly 4 times higher than values from UNTR tissue studied in similar glucose environments. Moreover, incubation of ENAL tissue in 20 mmol/L glucose caused a further 70% increase in SOD activity relative to 5 mmol/glucose conditions (P < 0.01 vs. UNTR in 20 mmol/L glucose). Thus, enhancement of SOD activity in renal cortex under these

·

Fig. 2. Effect of extracellular glucose concentration on O2− production by renal cortical slices from normal (UNTR) and enalapril-pretreated (ENAL) rats. Renal cortical slices were incubated at 37°C for 90 min in media containing either 5 mmol/L or 20 mmol/L glucose. Values are means ± SEM. *P < 0.05 vs. 5 mmol/L glucose within UNTR or ENAL groups, ‡P < 0.01 vs. UNTR at the same glucose concentration.

Fig. 3. Effect of extracellular glucose concentration on SOD activity in the renal cortex from normal (UNTR) and enalapril-pretreated (ENAL) rats. Renal cortical slices were incubated at 37°C for 90 min in media containing either 5 or 20 mmol/L glucose. Values are means ± SEM. *P < 0.05 vs. 5 mmol/L glucose within UNTR or ENAL groups, ‡P < 0.01 vs. UNTR at the same glucose concentration.

conditions is dependent on both acute exposure to elevated glucose levels and chronic enalapril treatment. NT content Fig. 4 shows the effect of chronic enalapril treatment on the NT content of renal cortical slices after incubation in media containing 5 or 20 mmol/L glucose. In the UNTR group, tissue NT content after incubation in media containing 20 mmol/L glucose averaged 5 times higher than after incubation in 5 mmol/L glucose media (P < 0.01). ENAL pretreatment did not

Fig. 4. Effect of extracellular glucose concentration on NT concentration in renal cortex from normal (UNTR) and enalapril-pretreated (ENAL) rats. Renal cortical slices were incubated at 37°C for 90 min in media containing either 5 or 20 mmol/L glucose. Values are means ± SEM. **P < 0.01 vs. 5 mmol/L glucose within UNTR or ENAL groups, ‡P < 0.01 vs. UNTR at the same glucose concentration.

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alter NT content in the normal glucose environment, but the stimulatory effect of 20 mmol/L glucose on NT content was completely suppressed in this group (P < 0.01 vs. the UNTR group in 20 mmol/L glucose; there was no significant difference between 5 and 20 mmol/L glucose within the ENAL group). Discussion We previously documented that renal afferent arteriolar diameter responses to NO synthase (NOS) inhibition are diminished in rats with DM and that SOD treatment (to diminish O2·− levels) restored the normal response to NOS inhibition in these animals [4]. Furthermore, SOD treatment restored the NO-dependent modulation of afferent arteriolar contractile responses to angiotensin II (AngII) in DM [26]. These observations implicate the interaction between O2·−, NO and AngII in regulation of renal vascular function in DM. NAD (P)H oxidase is emerging as an important source of vascular and renal O2·− production, and AngII is a potent stimulus of NAD(P) H oxidase activity. AngII levels in plasma (but not in kidney) are increased in the streptozotocin-induced model of DM in the rat, in concert with an increase in renal cortical AT1 receptor protein levels [27]. Thus, the beneficial effects of ACE inhibitors to slow progression of diabetic nephropathy may arise not only by virtue of a decrease in blood pressure achieved by control of AngII generation [12,17,18]. In fact, activation of an O2·− scavenging system and/or prevention of O2·− production per se by ACE inhibition may play a measurable role in the renoprotective effect on diabetic nephropathy with or without hypertension. The most fully characterized means of O2·− production is the cytokine-stimulated NADH oxidase reaction of neutrophils and macrophages, which forms massive amounts of O2·− with O2 consumption (2O2 + NAD(P)H + H+ 2O2·− + NAD + + − · (P) + 2H ). However, O2 can also be produced by NAD(P) H oxidase, xanthine oxidase and arachidonic acid metabolism systems that reside in multiple cell types, including vascular endothelial cells [12,28–33]. In recent reports on the multiple effects of ACE inhibitors, antioxidative effects involving the NAD(P)H oxidase system have been suggested to contribute to the beneficial effects of these agents in the kidney during DM [15,16,19]. The possibility exists that accelerated O2·− production is induced in vascular cells through protein kinase C (PKC)-dependent NAD(P)H oxidase activation during hyperglycemic conditions [30,31]. Moreover, Nishikawa et al. reported that generation of reactive oxygen species from mitochondrial sources is increased in the high-glucose condition [32]. Therefore, oxidative damage is considered as a potential pathogenic mechanism in diabetic nephropathy. In the present study, we clarified that NO and O2·− formation increases in the renal cortex under acute high-glucose conditions. The subsequent reaction of NO with O2·− is presumed to form ONOO−. NO is able to freely permeate the cell membrane by diffusion [2]. NO and O2·− react at a near diffusion-limited rate (k = 7 × 109 M−1 s−1) [7], and ONOO− is a more powerful cytotoxic factor compared to O2·−. SOD is the most efficient reduction catalyst and scavenger of O2·−

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(2O2·− + 2H+ → H2O2 + O2; k = 4 × 109 M−1 s−1). This reaction can be decelerated by half in the presence of physiological levels of chloride (k = 2 × 109 M−1 s−1) [8,34]. Consequently, the ONOO− formation rate is about 3-fold faster than O2·− scavenging by SOD [2,35]. The endogenous antioxidant enzyme SOD is abundant in various cells [36]; however, limitation of cell injury by the antioxidant action of SOD is insufficient in the pathophysiological conditions associated with supranormal production of O2·−. In the present study, renal cortical SOD activity was increased under the highglucose condition. Likewise, we previously reported that SOD activity was accelerated in the renal cortex of rats with DM [10]. We did not quantify SOD mRNA or protein levels in the present study. Sechi et al. reported a significant increase in Cu–Zn SOD expression in the kidney of rats with DM [37]. Moreover, enalapril augments activities of antioxidant enzymes in the kidneys of rats with DM [19,20]. The present study revealed that chronic enalapril treatment induced SOD activation and limited glucose-stimulated O2·− accumulation, which was interesting, although the underlying mechanism has not been clarified. In contrast with our result, glycosylation of SOD in DM causes inactivation of the enzyme [38,39]. The effect of chronic hyperglycemia to glycosylate SOD probably does not develop sufficiently during acute (90 min) exposure to highglucose levels, allowing other glucose-dependent processes that activate SOD to predominate under the conditions of our experiment. The effect of enalapril pretreatment on SOD activity implicates an AngII-dependent mechanism that modulates expression or activity of this enzyme and its responsiveness to the glucose environment. Notably, we were unable to detect an effect of 90 min of incubation in 5 vs. 20 mmol/L glucose media on renal cortical AngII content (0.9–1.2 fmol/g protein, unpublished observations). However, an effect of chronic enalapril treatment to depress renal and circulating AngII levels (and to increase kinin levels) may ultimately contribute to regulation of SOD activity. The availability of NOS substrate (L-arginine) can influence NO synthesis and may represent a critical determinant of whether the enzymatic product of NOS activity is NO (adequate substrate) or O2·− (limited substrate availability resulting in NOS uncoupling) [2,40,41]. We previously reported that a highglucose environment does not alter renal cortical L-arginine concentration measured under conditions identical to those employed in the present study [42]. Therefore, the glucoseinduced increase in renal cortical O2·− production cannot be attributed to NOS uncoupling due to limited substrate availability. A likely source of the glucose-induced O2·− production is NAD(P)H oxidase as AngII is a known stimulus of this enzyme and the present study demonstrated the ACEdependent nature of glucose-stimulated O2·− production. Although renal expression of NAD(P)H oxidase subunits (p47phox) are increased in DM [12], we do not know if 90 min of exposure to a high-glucose environment is capable of evoking this response. Thus, further studies are required to determine the possible role of NAD(P)H oxidase in the ability of acute glucose exposure to provoke O2·− production in the renal cortex.

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ONOO− is widely viewed as a pathophysiologic mediator [13,40]. Among the mechanisms through which ONOO− evokes cell damage is its ability to nitrate phenolic substances, especially tyrosine residues in protein. We previously utilized immunoblotting to demonstrate increased protein tyrosine nitration in the renal cortex of rats with DM [10]. The present study confirms (through the use of HPLC with electrochemical detection) the ability of an acute high-glucose environment to provoke an increase in renal cortical NT content. Protein tyrosine nitration may contribute to the development of diabetic nephropathy by provoking protein dysfunction (e.g. preventing tyrosine phosphorylation). Therefore, important determinants of cell injury by ONOO− include (1) co-localized and simultaneous O2·− and NO production in tissues, (2) the quantitative balance between O2·− and NO levels and (3) net protein NT levels. Kamisaki et al. showed that a 10-kDa molecular weight protein (nitrotyrosine denitrase) is able to extinguish NTcontaining protein, thus decreasing ONOO− toxicity [43]. The above considerations suggest that renoprotection in DM might be achieved by therapeutic control of NO and O2·− production (to limit ONOO− formation) or approaches that reverse tyrosine nitration. Recently, it has been reported that there is a renal protective action of angiotensin receptor blockers (ARB) as well as ACE inhibition [14,44–46]. The results of the present study indicate that ACE inhibition prevents glucose-stimulated oxidative stress and NT accumulation in the renal cortex, in part through effects to increase SOD activity (limit O2·− accumulation) and perhaps also by limiting O2·− production (minimizing AngII-dependent NAD(P)H oxidase activation). ACE inhibition may also influence nitrotyrosine denitrase activity, although little is known about regulation of this enzyme. In the future, it is necessary to further examine the potential antioxidant mechanism(s) that contribute to the renoprotective action of ARB and ACE inhibition during the early stage of DM. In summary, accelerated O2·− production and increased NT content in the renal cortex evoked by an acute high-glucose condition was completely prevented by enalapril treatment. These effects of enalapril were partly attributed to its enhancement of SOD activity. The increased NOX production was provoked by high glucose. Besides, high-glucose-induced NOX production was exaggerated by enalapril rather than ameliorated. These observations suggest that, apart from an anti-hypertensive effect, the renal protective action of ACE inhibition in DM might involve antioxidative effects including SOD activation and control of the renal cortex O2·− production per se. The functional influence of protein tyrosine nitration and its relationship to renal damage in DM remains to be clarified. Understanding of these processes may prove useful in developing diagnostic and therapeutic methods that can be employed during the early stage of DM to prevent or delay development of diabetic nephropathy. Acknowledgments The authors thank Yuji Hirowatari and Hideo Suzuki (Technology Department, Scientific Instruments Division,

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