Protective effect of apocynin, a NADPH-oxidase inhibitor, against contrast-induced nephropathy in the diabetic rats: A comparison with n-acetylcysteine

European Journal of Pharmacology 674 (2012) 397–406

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Pulmonary, Gastrointestinal and Urogenital Pharmacology

Protective effect of apocynin, a NADPH-oxidase inhibitor, against contrast-induced nephropathy in the diabetic rats: A comparison with n-acetylcysteine Akbar Ahmad a, 1, Stefania Mondello b, 1, Rosanna Di Paola c, Emanuela Mazzon c, Emanuela Esposito a, c, Maria Antonietta Catania a, Domenico Italiano c, Patrizia Mondello c, Carmela Aloisi c, Salvatore Cuzzocrea a, c,⁎ a b c

Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, Messina, Italy Department of Internal Medicine, University of Messina, Italy IRCCS Centro Neurolesi “Bonino-Pulejo”, Messina, Italy

a r t i c l e

i n f o

Article history: Received 6 June 2011 Received in revised form 17 October 2011 Accepted 30 October 2011 Available online 9 November 2011 Keywords: Apocynin NADPH-oxidase Contrast induced nephropathy

a b s t r a c t The aim of this study was to investigate the effects of apocynin, a NADPH (nicotinamide adenine dinucleotide phosphate)-oxidase inhibitor, in diabetic rats with nephropathy induced by contrast medium (CIN). Diabetes was induced in male Wistar rats by a single dose of streptozotocin (60 mg/kg i.v.). Animals were then divided into the following groups: 1) control group (diabetic rats treated i.v. with saline solution); 2) iomeprol group (iomeprol at 10 ml/kg was injected i.v. 30 min after saline administration); 3) apocynin group (identical to the iomeprol group, except for pre-treatment with apocynin 5 mg/kg i.v., 30 min before iomeprol injection) and 4) N-acetylcysteine group (NAC) (same as iomeprol group, except for the treatment with NAC 20 mg/kg i.v. 30 min before iomeprol injection). CIN in animals were assessed 24 h after administration of iomeprol. Apocynin significantly attenuates the impaired glomerular function, concentration of Na +, K +, alpha glutathione S-transferase levels in urine and neutrophil gelatinase-associated lipocalin levels in plasma caused by iomeprol. In kidney, immunohistochemical analysis of some inflammatory mediators, such as nitrotyrosine, poly-ADP-ribosyl polymerase, tumor necrosis factor-α, interleukin-1β as well as apoptosis (evaluated as terminal deoxynucleotidyltransferase-mediated UTP end labeling assay) revealed positive staining in tissue obtained from iomeprol group. These parameters were markedly reduced in animals treated with apocynin. Similarly, these parameters were also markedly modified by NAC pre-treatment. Here, we have shown that apocynin attenuates the degree of iomeprol-induced nephropathy in diabetic rats. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Contrast-induced nephropathy (CIN) is a complex syndrome of acute renal failure occurring after the administration of iodinated contrast media (McCullough and Soman, 2005). Diabetes mellitus seems to be independent risk factor in CIN and the incidence of CIN in diabetic patients varies from 5.7 to 29.4% (Nikolsky et al., 2004). In an attempt to assess the influence of diabetic and pre-diabetic state on the development of CIN in chronic kidney disease patients, it was demonstrated that patients with diabetes mellitus are at a higher risk of developing CIN, but patients with pre-diabetes mellitus are not at as high a risk for developing CIN as diabetes patients

⁎ Corresponding author at: Department of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Torre Biologica, Policlinico Universitario Via C. Valeria, Gazzi, 98100 Messina, Italy. Tel.: + 39 090 2213644; fax: + 39 090 2213300. E-mail address: [email protected] (S. Cuzzocrea). 1 The authors contributed equally to this work. 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.10.041

(Toprak et al., 2007). CIN has become the third leading cause of hospital acquired acute renal failure, accounting for 10 to 25% of all acute renal failure cases, also considering the increasing use of contrast media in diagnostic and interventional procedures (Finn, 2006; Goldenberg and Matetzky, 2005; Mehran and Nikolsky, 2006; Tepel et al., 2006). The exact mechanism of CIN is not fully elucidated. Based on several studies, oxygen radicals play a major causative role as the primary physiological insult (Bakris et al., 1990; Bakris et al., 1999; Baliga et al., 1997; Heyman et al., 1999; Parvez et al., 1989; Yoshioka et al., 1992). Infusion of contrast agents increases osmotic load, viscosity, hypoxemia of the renal medulla and renal free radical production through post-ischemic oxidative stress (Brezis and Rosen, 1995; Katholi et al., 1998). This is due to decreased tissue oxygen tension, which promotes mitochondria generation of reactive oxygen species (Chandel et al., 2000; Dada et al., 2003). Superoxide anion (O2−) and perhaps reactive oxygen species have been discussed to promote CIN. Oxygen radicals are endogenously produced and levels can increase during oxidative stress. The most common oxygen radicals are O2−, hydrogen peroxide (H2O2) and hydroxyl radical (OH −)

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(Schnackenberg, 2002). O2− and OH − are more reactive than H2O2, which is not a radical, but exhibits greater membrane permeability. O2− rapidly scavenges nitric oxide (NO) and could therefore blunt NO activity in the renal microvascular. Since NO inhibits oxygen consumption, it is tempting to speculate that reduced (scavenged) NO during diabetes elevates oxygen consumption, thereby leading to reduced partial oxygen pressure values with consequences for endothelial–epithelial structure and function. Reactive oxygen species may play a role in the effects of various vasoconstrictors that have been considered important for the development of CIN. Apocynin (4-hydroxy-3-methoxy-acetophenone) is a constituent of the Himalayan herb Picrorhiza kurroa Royle (Scrophulariaceae) that is well known in traditional Indian medicine (Ayurveda). It is an acetophenone to which a range of biological activities is attributed (Hougee et al., 2006). It is a prodrug that is converted by peroxidasemediated oxidation to a dimer, which has been shown to be more efficient than apocynin itself (Johnson et al., 2002). It has been used as an efficient inhibitor of the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase in many experimental model such as colitis, rheumatoid arthritis, ischemia reperfusion and lung injury (Dodd-o et al., 2004). Based on these observations, this study was to investigate the potential therapeutic role of apocynin, a reversible inhibitor of NADPH oxidase, on contrast medium-induced nephropathy in diabetic rats. 2. Materials and methods 2.1. Animals Male Wistar rats (150–200 g; Harlan Nossan; Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purposes (D.M.116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986). 2.2. Induction of experimental diabetes After 12 h of fasting, the animals received a single 60 mg/kg intravenous injection of streptozotocin (Sigma, St. Louis, MO) in 10 mM sodium citrate buffer, pH 4.5 (see figure). After 24 h, animals with blood glucose levels greater than 250 mg/dl were considered diabetic. We performed all of experiments with contrast medium 10 days following the induction of diabetes. We confirmed the diabetic state a evaluating daily the blood glucose levels. 2.3. Contrast-induced nephropathy (CIN) Male diabetic rats were randomly allocated into the following groups: (1) Control group. Rats were treated intravenously (i.v.) with saline solution (n = 5); (2) Iomeprol group. Rats were administered iomeprol (10 ml/kg i.v.) 30 min after saline administration (n = 5); (3) Apocynin group. Identical to the Iomeprol group, except for the administration of apocynin (5 mg/kg i.v.) instead of saline administration (n = 5); (4) N-acetylcysteine (NAC) group. Identical to Iomeprol group, except for the administration of NAC (20 mg/kg i.v.) instead of saline administration (n = 5). In this experiment, the dose of apocynin was chosen in agreement with our previous studies (Genovese et al., 2011; Impellizzeri et al., 2011; Paterniti et al., 2010). 2.4. Measurement of biochemical parameters After 24 h, the urine samples were collected and blood samples were obtained via the lateral tail vein into S1/3 tubes containing serum gel. The samples were centrifuged (6000 g for 3 min) to separate

plasma. All plasma samples were analyzed for biochemical parameters within 24 h after collection. Concentrations of urea and creatinine in plasma and urine were measured as indicators of impaired glomerular function. Creatinine clearance (ml/min) was calculated using the following formula (=UV/P), where U refers to creatinine concentration in urine (mg/dl), V to urine volume/min (ml/min) and P to serum creatinine (mg/dl). A kidney injury biomarker called “Neutrophil gelatinase-associated lipocalin” (NGAL) was evaluated as indicated by the commercial kit in plasma and urine. Urine concentration of αGST as well as urine and plasma concentration of NGAL were evaluated as indicated by the commercial kit. Na+ and K + concentrations in plasma were determined by flame photometry. 2.5. Histological evaluation Twenty-four hours after administration of saline or apocynin or NAC, animals were sacrificed and a 5 μm section of kidney was removed and placed in formalin and processed through to wax. Five millimeter sections were cut and stained with hematoxylin and eosin. Histological assessment of outer medulla damage was examined by an experienced morphologist, who was not aware of the sample identity. The criteria for injury/necrosis were the following: 0 = normal histology; 1 = minor edema, minor cell swelling; 2 = hemorrhage, moderate edema, moderate cells vacuolization and swelling; 3 = moderate hemorrhage, moderate edema, moderate cells vacuolization, swelling and chromatin alteration; 4 = severe edema, severe cells vacuolization, swelling and chromatin alteration, presence of necrosis spot; 5 = severe edema, severe cytoplasmic vacuolar changes, swelling and chromatin alteration, intratubular cast formation, renal tubular architecture, luminal congestion and severe necrosis. 2.6. Immunohistochemical localization of nitrotyrosine, peroxisome proliferators-activated receptors (PAR), tumor necrosis factor (TNF)-α, interleukin (IL)-1β At the end of experiment, tissues were fixed in 10% (w/v) PBS-buffered formalin and 8 μm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% (v/v) hydrogen peroxide in 60% (v/v) methanol for 30 min. The sections were permeabilized with 0.1% (v/v) Triton X-100 in PBS for 20 min. Non-specific adsorption was minimized by incubating the section in 2% (v/v) normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin (Vector Laboratories, Burlingame, CA). The sections were then incubated overnight with 1:1000 dilution of primary anti-nitrotyrosine antibody (Millipore, 1:500 in PBS, v/v), anti-poly (ADP)-ribose (PAR) antibody (Santa Cruz Biotechnology, 1:500 in PBS, v/v), anti-TNF-α antibody (Santa Cruz Biotechnology, 1:500 in PBS, v/v), anti-IL-1β antibody (Santa Cruz Biotechnology, 1:500 in PBS, v/v). Controls included buffer alone or non-specific purified rabbit Immunoglobulin G. Specific labeling was detected with a biotin-conjugated specific secondary anti-IgG and avidin–biotin peroxidase complex (Vector Laboratories, Burlingame, CA). In order to confirm that the immunoreactions for the nitrotyrosine were specific, some sections were also incubated with the primary antibody (anti-nitrotyrosine) in the presence of excess nitrotyrosine (10 mM) to verify the binding specificity. To verify the binding specificity for nitrotyrosine, PARP, TNF-α and IL-1β, some sections were also incubated with primary antibody only (no secondary antibody) or with secondary antibody only (no primary antibody). In these situations, no positive staining was found in the sections indicating that the immunoreactions were positive in all the experiments carried out. Immunocytochemistry photographs (n = 5) were assessed by densitometry. The assay was carried out by using Optilab Graftek software on a Macintosh personal computer (CPU G3-266). All the

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immunocytochemistry analysis was carried out without knowledge of the treatments. 2.7. Terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) assay TUNEL assay was conducted by using a TUNEL detection kit according to the manufacturer's instructions (Apotag, HRP kit DBA, Milan, Italy). Briefly, sections were incubated with 15 μg/ml proteinase K for 15 min at room temperature and then washed with PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at room temperature and then washed with PBS. Sections were immersed in terminal deoxynucleotidyltransferase and biotinylated dUTP in TdT buffer, incubated in a humid atmosphere at 37 °C for 90 min, and then washed with PBS. The sections were incubated at room temperature for 30 min with anti-horseradish peroxidaseconjugated antibody, and the signals were visualized with diaminobenzidine. The number of TUNEL positive cells/high-power field was counted in 5–10 fields for each coded slide. 2.8. Materials Unless otherwise stated, all compounds were obtained from Sigma-Aldrich Company Ltd. (Milan, Italy). All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl; Baxter, Italy) or 10% dimethyl sulfoxide (DMSO). 2.9. Statistical evaluation All values in the figures and text are expressed as mean ± standard error of the mean (S.E.M.) of N observations. For the in vivo studies N represents the number of animals studied. In the experiments involving histology, the figures shown are representative of at least three experiments performed on different experimental days. The results were analyzed by one-way ANOVA followed by a Bonferroni post hoc test for multiple comparisons. A P-value of less than 0.05 was considered significant. 3. Results 3.1. Effect of iomeprol on kidney function in non diabetic or diabetic rats In this preliminary study, effect of vehicle on non diabetic and diabetic rats, no significant alteration in plasma concentration of urea (1A) and creatinine (Fig. 1B) was found. To study the clinical situation of CIN, non diabetic (Sham animals) or diabetic rats were sacrificed at 24 h after the administration of the contrast agent iomeprol at 10 ml/kg i.v. In this, a significant increase in the plasma concentrations of urea (Fig. 1A) and creatinine (Fig. 1B) was observed in diabetic rats at 24 h after the administration of iomeprol. On the contrary, no alteration of the plasma concentrations of urea (Fig. 1A) and creatinine (Fig. 1B) was observed in non diabetic rats at 24 h after the administration of iomeprol. Similarly, the treatment of non diabetic rats with NAC (10 mg/kg) and apocynin (5 mg/kg) did not produced significant alteration in plasma urea and creatinine (data not shown) as previously demonstrated (research paper on zymosan, NAC and apocynin). Based on these findings, all further experiments were performed on diabetic rats at 24 h after the administration of iomeprol. 3.2. Effect of apocynin and NAC on CIN-mediated glomerular dysfunction In the rat model of CIN at 24 h the animals exhibited significant elevations in plasma concentrations of urea (Fig. 2A), creatinine (Fig. 2B), as well as a significant decrease in creatinine clearance (Fig. 2C) compared with control diabetic rats (Fig. 2A, B, C). Pretreatment with a single intravenous bolus of apocynin (5 mg/kg) at

Fig. 1. Effect of vehicle and iomeprol on kidney function in non diabetic or diabetic rats. No significant increase in the plasma concentrations of urea (A) and creatinine (B) was observed in non-diabetic and diabetic rats at 24 h after the administration of saline. In addition, at 24 h after injections of iomeprol diabetic rats showed an important and significant increase in plasma concentrations of urea (A) and creatinine (B) as compared to non diabetic rats. The figures are representative of at least three experiments performed on different experimental days. Each data are expressed as Mean ± S.E.M. from n = 5 male Wistar rats for each group. A P-value of less than 0.05 was considered significant. *P b 0.01 vs streptozotocin + iomeprol.

30 min before the injection of iomeprol significantly attenuated the iomeprol-induced glomerular dysfunction in comparison with the iomeprol group (Fig. 2A, B, C). Similarly, administration of NAC (20 mg/kg, i.v.) significantly improved the iomeprol-induced glomerular dysfunction in comparison with the iomeprol group (Fig. 2A, B, C).

3.3. Effect of apocynin and NAC on Na + and K + in plasma levels at 24 h after CIN induction In this study we show that no significant alteration in plasma concentrations of Na + (Fig. 3A) and K + (Fig. 3B) was observed in control diabetic rats at 24 h after the administration of saline in comparison to non diabetic animals (data not shown). In addition an important and significant increase in plasma concentrations of Na + (Fig. 3A) as well as a decrease in concentrations of K + (Fig. 3B) were observed in diabetic rats at 24 h after the injection of the iomeprol. Pre-treatment with a single intravenous bolus of apocynin (5 mg/kg) produced a significant reduction of plasma Na+ (Fig. 3A) as well as a significantly restored plasma concentrations of K + (Fig. 3B). Similarly, the treatment with NAC (20 mg/kg) produced a significant reduction

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Fig. 2. Effect of apocynin and NAC on CIN-mediated glomerular dysfunction. No significant increase in the plasma concentrations of urea (A) and creatinine (B) was observed in diabetic rats at 24 h after the administration of saline in comparison to non diabetic animals (data not shown). In addition, at 24 h after injections of iomeprol diabetic rats showed an important and significant increase in plasma concentrations of urea (A) and creatinine (B) as well as a decrease in creatinine clearance (C) was observed. The treatment with apocynin reduced plasma urea (A), creatinine (B) as well as increased creatinine clearance (C). The same improvement in glomerular dysfunction was observed in rats treated with NAC. The figures are representative of at least three experiments performed on different experimental days. Each data are expressed as Mean ± S.E.M. from n = 5 male Wistar rats for each group. A P-value of less than 0.05 was considered significant. *P b 0.01 vs streptozotocin, °P b 0.01 vs streptozotocin + iomeprol.

in plasma Na + (Fig. 3A) as well as a significantly increased plasma concentration of K+ (Fig. 3B).

3.4. Effect of apocynin and NAC on plasma and urine NGAL levels In this study we observed a significant increase in plasma and urine levels of NGAL in animals from iomeprol group (Fig. 4A) compared with control group (Fig. 4A) at 24 h. Pre-treatment with apocynin (5 mg/kg) produced a significant reduction in plasma and urine NGAL (Fig. 4A and B) levels. Similarly, the treatment with NAC (20 mg/kg) produced a significant reduction in plasma (Fig. 4A) and urine NGAL (Fig. 4B) concentrations at 24 h after the injection of the iomeprol.

Fig. 3. Effect of apocynin and NAC on Na+ and K+ in plasma levels at 24 h after CIN induction. No significant alteration in the plasma concentrations of Na+ (A) and K+ (B) was observed in diabetic rats at 24 h after the administration of saline in comparison to non diabetic animals (data not shown). In addition, diabetic rats after iomeprol showed a significant increase in plasma concentrations of Na+ (A) as well as a significantly lower concentration of K+ (B). The treatment with apocynin produced significant reduction in plasma Na+ (A) and restored plasma concentrations of K+ (B). Similarly, the treatment with NAC produced a significant reduction in plasma Na+ (A) as well as an important increase in plasma concentrations of K+ (B). The figures are representative of at least three experiments performed on different experimental days. Each data are expressed as Mean± S.E.M. from n = 5 male Wistar rats for each group. A P-value of less than 0.05 was considered significant. *P b 0.01 vs streptozotocin, °P b 0.01 vs streptozotocin + iomeprol.

3.5. Effect of apocynin and NAC on urine αGST levels A significant increase in the urine concentrations of αGST, a sensitive and specific assay for proximal injury assessment (Fig. 4C), was observed in diabetic rats at 24 h after the administration of saline in comparison to non diabetic animals. The treatment with NAC (20 mg/kg) as well as with apocynin (5 mg/kg) produced a significant reduction in urine αGST (Fig. 4C) concentrations at 24 h after the administration of the contrast agent. 3.6. Effect of apocynin and NAC on CIN-mediated renal histopathology The iomeprol group presented marked alternations in renal histology compared to control group (Fig. 5A and B, see histological score

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markedly diminished histologic features (Fig. 5C and D, see histological score E). 3.7. Effect of apocynin and NAC on CIN-mediated nitrotyrosine formation and PARP activation Immunohistochemical analysis of kidney sections obtained from diabetic rats treated with iomeprol revealed positive staining for nitrotyrosine (Fig. 6A, see densitometry analysis Fig. 6I). In contrast, reduced staining for nitrotyrosine was found in the kidney from iomeprol-treated diabetic rats which had been treated with apocynin (Fig. 6C, see densitometry analysis Fig. 6I). Similarly, the treatment with NAC reduced the degree of positive staining for nitrotyrosine (Fig. 6D, see densitometry analysis I). At the same time point (24 h after iomeprol administration), kidney tissue sections were taken in order to determine the immunohistological staining for poly ADPribosylated proteins (an indicator of PARP activation). A positive staining for the PAR (Fig. 6F, see densitometry analysis Fig. 6I) was found primarily localized in the inflammatory cells present in the kidney tissue from iomeprol-treated diabetic rats. Apocynin treatment reduced the degree of PARP activation (Fig. 6G, see densitometry analysis Fig. 6I). Similarly, the treatment with NAC reduced the degree of PARP activation (Fig. 6H, see densitometry analysis I). Please note that there was no staining for either nitrotyrosine (Fig. 6A, see densitometry analysis Fig. 6I) or PAR (Fig. 6E, see densitometry analysis Fig. 6I) in kidney tissues obtained from control group diabetic rats. 3.8. Effect of apocynin and NAC on CIN-mediated pro-inflammatory cytokine release in the kidney

Fig. 4. Effect of apocynin and NAC on plasma and urine NGAL levels and urine α-GST levels. No significant increase in the plasma and urine levels of NGAL (A and B) was observed in diabetic rats at 24 h after the administration of saline in comparison to non diabetic animals (data not shown). In addition an important and significant increase in plasma and urea levels of NGAL (A and B) was observed in diabetic rats at 24 h after injection of iomeprol. The treatment with apocynin produced a significant reduction in plasma and urea NGAL (A and B) levels at 24 h after the administration of iomeprol. Similarly, the treatment with NAC produced a significant reduction in plasma and urea NGAL (A and B) levels at 24 h after the administration of iomeprol. No significant increase in urine levels of alpha-GST (C) was observed in diabetic rats at 24 h after the administration of saline in comparison to non diabetic animals (data not shown). In addition an important and significant increase in urea levels of alpha-GST (C) was observed in diabetic rats at 24 h after injection of iomeprol. The treatment with apocynin produced a significant reduction in urea alpha-GST (C) levels at 24 h after the administration of iomeprol. Similarly, the treatment with NAC produced a significant reduction in urea alpha-GST (C) levels at 24 h after the administration of iomeprol. The figures are representative of at least three experiments performed on different experimental days. Each data are expressed as Mean ± S.E.M. from n = 5 male Wistar rats for each group. A P-value of less than 0.05 was considered significant. *Pb 0.01 vs streptozotocin, °P b 0.01 vs streptozotocin + iomeprol.

E). The outer stripe of the proximal modulated tubule cells was affected in the renal zones by the contrast medium. Specifically, the most severe and pronounced alterations were observed in the renal tubular architecture, including severe cytoplasmic vacuolar changes, intratubular cast formation, and luminal congestion (Fig. 5B see histological score E). Kidney sections from iomeprol group that were pretreated with apocynin (5 mg/kg) or NAC (20 mg/kg) demonstrated

In this study we have evaluated TNF-α and IL-β expression in the kidney tissues by immunohistochemical analysis. Tissue sections obtained from diabetic rats at 24 h after iomeprol injection demonstrate positive staining for TNF-α mainly localized in the infiltrated inflammatory cells, pneumocytes as well as in vascular wall (Fig. 7B, see densitometry analysis Fig. 7I). In contrast, no staining for TNF-α was found in the kidneys from iomeprol-injected rats that have been treated with apocynin (Fig. 7C, see densitometry analysis Fig. 7I). Similarly, iomeprol-injected diabetic rats treated with NAC showed reduced degree of staining for TNF-α (Fig. 7D, see densitometry analysis Fig. 7I). At the same time point (24 h after iomeprol administration), kidney tissue sections were taken in order to determine the immunohistological staining for IL-1β. A positive staining for IL-1β mainly localized in the infiltrated inflammatory cells was observed in kidney tissue sections obtained from iomeprol-injected diabetic rats (Fig. 7F, see densitometry analysis Fig. 7I). Apocynin treatment reduced the degree of IL-1β expression (Fig. 7G, see densitometry analysis Fig. 7I). Similarly, the diabetic rats treated with NAC showed a lower degree of IL-β expression (Fig. 7H, see densitometry analysis Fig. 7I). Please note that there was no staining for either TNF-α (Fig. 7A, see densitometry analysis Fig. 7I) or IL-β (Fig. 7E, see densitometry analysis Fig. 7I) in kidney tissues obtained from control group diabetic rats. 3.9. Effect of apocynin and NAC on apoptosis To investigate whether CIN is associated with apoptotic cell death we measured TUNEL-like staining in kidney tissues. At 24 h after iomeprol administration, kidney tissue demonstrated a marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments in both glomerular as well as tubules (Fig. 8A and C see Fig. 8E). In contrast, no apoptotic cells or fragments were observed in the tissues obtained from iomeprol-injected diabetic rats treated with apocynin (Fig. 8B see Fig. 8E). Similarly, no apoptotic cells or fragments were observed in the tissues obtained from iomeprolinjected diabetic rats that were treated with NAC.

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Fig. 5. Effect of apocynin and NAC on CIN-mediated renal histopathology. No histological alterations were observed in the kidney from Sham group (A). In addition, a severe kidney injury was observed in iomeprol group after 24 h (B, see histological score E). The treatment with apocynin (C, see histological score E) significantly reduced the degree of kidney injury at 24 h. Similarly, the treatment with NAC produced a significant reduction of the kidney injury at 24 h (D, see histological score E). The figures are representative of at least three experiments performed on different experimental days. Each data are expressed as Mean ± S.E.M. from n = 5 male Wistar rats for each group. A P-value of less than 0.05 was considered significant. *P b 0.01 vs streptozotocin, °P b 0.01 vs streptozotocin + iomeprol.

4. Discussion In this report, we investigated the effects of apocynin, an NADPH inhibitor, on CIN in diabetic rats. We demonstrate that apocynin attenuated: (i) glomerular dysfunction, (ii) Na + and K + levels in plasma, (iii) alpha GST levels in urine and NGAL levels in plasma as well as urine, (iv) the degree of kidney injury caused by iomeprol administration, (v) nitrotyrosine formation and PARP activation, (vi) pro-inflammatory cytokines production, and (vii) apoptosis. All of these findings support the view that apocynin markedly reduced the degree of CIN in diabetic rats. What, then, is the mechanism by which apocynin reduces iomeprol induced nephropathy in diabetic rats? Apocynin is an efficient inhibitor of NADPH oxidase, a functional enzyme that generates reactive oxygen species during the inflammatory process not as a byproduct, but rather as the primary function of the enzyme system (Bedard and Krause, 2007). Activation of NADPH-oxidase is associated with the migration of the cytosolic components to the cell membrane so that the complete oxidase can be assembled (Smit et al., 2000). Apocynin prevents the translocation of p47phox to Nox2 in leukocytes, monocytes, and endothelial cells (Johnson et al., 2002; Stolk et al., 1994). It is assumed that apocynin is activated by H2O2 and MPO to form an apocynin radical, which then oxidizes thiols in the NADPH-oxidase. Indeed, thiols are critical for the function of p47phox, and thiol oxidizing agents have been shown to block NADPH-oxidase activation (Clark et al., 1990; Johnson et al., 2002). In diabetic nephropathy endothelial dysfunction of renal vessels with suppressed NO activity in the renal microvasculature is frequent (Schnackenberg, 2002). Superoxide production is indeed enhanced in renal cortical tissue from diabetic rats (Ishii et al., 2001) along with a diminished afferent and efferent arteriolar vasoconstrictor response to NOS inhibition (Palm et al., 2003). Thus it seems that superoxide, and/or further reactive oxygen species may be involved in CIN. The pathogenic mechanism of CIN is controversial, the direct toxic effect of contrast medium on the renal tubular cells is known to play an important role in its development (Goldenberg and Matetzky, 2005). Disturbance in renal hemodynamic and direct

cytotoxicity have been identified as key factors in the pathogenesis of CIN. Low oxygen tension normally exists on the outer renal medullar region, reflecting the sensitive regional oxygen supply and a high local metabolic rate and oxygen requirement, resulting from active salt reabsorption by medullar thick ascending limbs of Henle's loop. Contrast agents markedly aggravate this physiologic hypoxia of the outer medullar layer because they cause enhanced metabolic activity and oxygen consumption as a result of osmotic diuresis and increased salt delivery to the distal nephron. The result of the hemodynamic changes is hypoxia followed by oxidative stress and repair. In this study we revealed that pre-treatment with apocynin is sufficient to completely prevent renal deterioration and improve renal function. NGAL seems to have more complex activities than its anti-microbial effect. The expression of NGAL rises 1000-fold in humans and rodents in response to renal tubular injury, and it appears so rapidly in the urine and serum that it is useful as an early biomarker of renal failure (Schmidt-Ott et al., 2006). In present study we have observed the significant increase in plasma as well as urine NGAL concentrations in CIN diabetic animals but pre-treatment with apocynin (5 mg/kg) produced a significant reduction in plasma as well as in urine NGAL concentrations 24 h after the administration of the iomeprol. Urinary levels of alpha-glutathione S-transferase (alpha-GST) markers of proximal and distal renal tubule damage, in our study we found significant increase in urine alpha-GST concentrations in CIN diabetic animals but pre-treatment with apocynin (5 mg/kg) produced a significant reduction in urine alpha-GST concentrations 24 h after the administration of the iomeprol. The most severe and pronounced alterations were observed in the renal tubular architecture, including severe cytoplasmic vacuolar changes, intratubular cast formation, and luminal congestion. The kidney sections from iomeprol group that were pre-treated with apocynin demonstrated markedly diminished histological changes. Several studies demonstrated that apocynin significantly inhibited the expression of TNF-α and IL-1β which are potent triggers involved in leukocyte migration (Crofford et al., 1997) in arthritic animals. There is evidence that the pro-inflammatory cytokines TNF-α and IL-1β help to propagate the extension of a local or systemic inflammatory

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Fig. 6. Effect of apocynin and NAC on nitrotyrosine formation and PARP activation. Sham group having no positive staining for nitrotyrosine (A) and PAR (F) in kidney tissue section. CIN group shows positive staining for nitrotyrosine (B) as well as PAR (G), these were mainly localized on inflammatory cells. Apocynin treatment reduced the degree of positive staining for nitrotyrosine (C) and PAR (H) in the tissues sections. Similarly, treatment with NAC reduced the degree of positive staining for nitrotyrosine (D) and PAR (I) in tissues sections. Densitometry analysis of immunocytochemistry photographs for nitrotyrosine (E) and PAR (J) were assessed. The figures are representative of at least three experiments performed on different experiment day. Each data are expressed as Mean ± S.E.M. from n = 5 male wistar rats for each group. A P-value of less than 0.05 was considered significant. *P b 0.01 vs streptozotocin, °P b 0.01 vs streptozotocin+iomeprol.

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Fig. 7. Effect of apocynin and NAC on CIN-mediated pro-inflammatory cytokine release in the kidney. No positive staining for TNF-α (A) and IL-1β (F) was found in kidney tissue section from Sham group. In tissue sections obtained from iomeprol group positive staining for TNF-α (B) and for IL-1β (G) were mainly localized in inflammatory cells. The treatment with apocynin reduced the degree of positive staining for TNF-α (C) and for IL-1β (H) in the kidney tissues. Similarly, the treatment with NAC reduced the degree of positive staining for TNF-α (D) and for IL-1β (I) in the kidney tissues. Densitometry analysis of immunocytochemistry photographs for TNF-α (E) and for IL-1β (J) from kidney tissues assessed. The figures are representative of at least three experiments performed on different experimental days. Each data are expressed as Mean ± S.E.M. from n = 5 male Wistar rats for each group. A P-value of less than 0.05 was considered significant. *P b 0.01 vs streptozotocin, °P b 0.01 vs streptozotocin + iomeprol.

process (Volman et al., 2002). We confirm here that iomeprol induced nephropathy leading to a substantial increase in the levels of both TNF-α and IL-1β in the plasma after 24 h. Treatment of iomeprol-

injected diabetic rats with apocynin attenuated the production of TNF-α and IL-1β. These data are confirmed by immunohistochemical localization of these cytokines. Indeed, the assessment of kidney tissue

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Fig. 8. Effect of apocynin and NAC on apoptosis. At 24 h after iomeprol administration, kidney tissue demonstrated a marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments in both glomerular as well as tubules (A and C see panel E). In contrast, no apoptotic cells or fragments were observed in the tissues obtained from iomeprol-injured diabetic rats treated with apocynin (D see panel E). Similarly, no apoptotic cells or fragments were observed in the tissues obtained from iomeprol diabetic rats with NAC (Fig. 8B see Fig. 8E). The figures are representative of at least three experiments performed on different experimental days. Each data are expressed as Mean ± S. E.M. from n = 5 male Wistar rats for each group. A P-value of less than 0.05 was considered significant. *P b 0.01 vs streptozotocin, °P b 0.01 vs streptozotocin + iomeprol.

sections have revealed a higher expression of TNF-α and IL-1β in samples obtained from iomeprol-injected diabetic rats, while apocynin treatment exhibited lower cytokines staining (Volman et al., 2002). Nitrotyrosine formation, along with its detection by immunostaining, was initially proposed as a relatively specific marker for the detection of the endogenous formation of peroxynitrite and an increased nitrotyrosine staining is considered as an indication of increased nitrosative stress (Di Paola et al., 2006). Thus, by immunohistochemical localization, we showed an increase in nitrotyrosine staining in kidney obtained from iomeprol-induced nephropathy in diabetic rats, while an improvement was due to apocynin administration. There is a large amount of evidence that reactive oxygen species promote CIN (Qin et al., 1999). A novel pathway of inflammation, governed by the nuclear enzyme PARP has been proposed in relation to hydroxyl radical- and peroxynitrite-induced DNA single strand breakage. This pathway plays an important role in various models of inflammation as well as in iomeprol-induced nephropathy (Cuzzocrea et al., 1997). We demonstrated here that apocynin attenuates the increase in PARP activity in the kidney from iomeprol-injected diabetic rats. Thus, we propose that the effect of apocynin may be at least in part due to the prevention of the activation of PARP. In this study we investigated apoptotic cell death by TUNEL-like staining in kidney tissues. At 24 h after iomeprol injection, kidney tissue demonstrated a marked appearance of dark brown apoptotic cells and intercellular apoptotic fragments in both glomerular as well as tubules. In contrast, no apoptotic cells or

fragments were observed in the tissues obtained from iomeprolinjected diabetic rats treated with apocynin. Recent studies have also shown that inhibition of NADPH-oxidase by apocynin reduces cardiomyocyte apoptosis in response to angiotensin II (Qin et al., 2006). This means that the apocynin by inhibiting NF-κB prevents the loss of the anti-apoptotic way and reduced the pro-apoptotic pathway activation with a mechanism still to discover.

5. Conclusion Our evidence has shown that intravenous N-acetylcysteine as well as apocynin are safe and effective in preventing CIN in diabetic rats. Further studies are needed in order to clarify the valid mechanism underlying this effect.

Acknowledgments The authors would like to thank Giovanni Leotta, Carmelo La Spada for their excellent technical assistance during this study, Mrs Caterina Cutrona for secretarial assistance and Miss Valentina Malvagni for editorial assistance with the manuscript. This study was supported by a grant from IRCCS Centro Neurolesi “Bonino-Pulejo”.

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