http://informahealthcare.com/rnf ISSN: 0886-022X (print), 1525-6049 (electronic) Ren Fail, 2014; 36(5): 774–780 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2014.884396
LABORATORY STUDY
Inhibition of inducible nitric oxide synthase prevents shock wave therapy induced renal injury Bilal Firat Alp1, Ercan Malkoc2, Zafer Demirer1, Ali Guragac1, Turker Turker3, Ertan Altayli4, Ayhan Ozcan5, Bulent Uysal6, Turgut Topal6, Emin Ozgur Akgul7, Ibrahim Yildirim1, and Ahmet Guven8 1
Department of Urology, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, 2Division of Urology, Military Hospital, Tekirdag, Turkey, Department of Epidemiology, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, 4Department of Research and Development Center, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, 5Department of Pathology, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, 6Department of Physiology, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, 7Department of Biochemistry, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, and 8Department of Pediatric Surgery, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey
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Abstract
Keywords
Objectives: Shock wave lithotripsy treatment (SWT) is not completely free from side effects; one of the accused mechanisms for renal injury during SWT is oxygen- and nitrogen-derived free radical productions. Therefore, we aimed to evaluate the effect of inhibition of nitric oxide (NO) production by N-[3(aminomethyl) benzyl) acetamidine] (1400W), highly selective inducible nitric oxide synthase (iNOS) inhibitor, at SWT-induced kidney damage. Materials and methods: Twenty-four rats those underwent right nephrectomy procedure were divided equally into three groups as control, SWT, and SWT + 1400W. 1400W was administered at a dose of 10 mg/kg at 2 h prior to SWT procedure and at the beginning of SWT procedure via intraperitoneal route and continued daily for consecutive 3 days. At the end of the fourth day, animals were killed via decapitation and trunk blood and the left kidneys were taken for biochemical and histopathologic evaluation. Results: SWT caused renal tubular damage and increased lipid peroxidation and antioxidant enzyme activities and SWT also significantly increased nitro-oxidative products. Inhibition of iNOS via administration of 1400W ameliorated renal injury and decreased tissue lipid peroxidation (malondialdehyde), superoxide dismutase, glutathione peroxidase and nitrite/nitrate levels (NOx). In addition, it was seen that histolopathological changes were attenuated in the SWT + 1400W group when compared to SWT group. Conclusion: SWT-induced renal injury might be due to excessive production of oxygen free radicals and NO production. Inhibition of iNOS attenuates renal injury following SWT treatment. It can be concluded that iNOS inhibitors or peroxynitrite scavengers might be used to protect the kidneys against SWT-induced morphological and functional injuries.
1400W, iNOS inhibitor, oxidative stress, renal injury, shock wave therapy
Introduction Since its first presentation, extracorporeal shock wave lithotripsy treatment (SWT) has gained popularity in treating kidney stones because of its ease of use, noninvasive nature and high efficacy. However, despite its marked effectiveness, SWT is not completely free from side effects. The main target of the shockwaves applied during a SWT procedure are stones located in the kidney but the surrounding tissues, renal parenchyma, and other organs are traumatized during this procedure.1 For the last two decades, more reports evaluating the adverse effects of SWT with experimental as well as clinical
Address correspondence to Ahmet Guven, Department of Pediatric Surgery, Gulhane Military Medical Faculty, 06017 Etlik, Ankara, Turkey. Tel.: +90 312 3045484; Fax: +90 312 3042150; E-mail: [email protected]
History Received 10 September 2013 Revised 9 December 2013 Accepted 28 December 2013 Published online 10 February 2014
studies came out.2,3 Concerning the mechanism of the adverse effects of SWT, reports have suggested that, renal damage is induced by the physical force of the waves and transient ischemia caused by both local and regional vasoconstriction, and intra-parenchymal bleeding.4,5 As a result of these insults, release of cytokines/inflammatory cellular mediators, production of oxygen-derived [reactive oxygen species (ROS)] free radicals, such as superoxide (O 2 ), peroxide (H2O2) and hydroxyl radicals (OH), production of nitrogen-derived free radicals (reactive nitrogen species), such as nitric oxide (NO) peroxynitrite (ONOO), and infiltration of tissue by inflammatory response cells initiate the process of cellular damage and death.6 However, this hypothesis is supported by researches that a variety of antioxidant agents have been found to be effective in SWT induced renal parenchymal injury.7–9 Nevertheless, the biochemical mechanisms underlying tissue damage have yet to be fully elucidated. One of the mechanism discussed for tissue damage in SWT is free oxygen radical production.2 Hydroxyl radicals and
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DOI: 10.3109/0886022X.2014.884396
superoxide anions are well known oxygen free radicals, which impose cytotoxic effects on cells. Moreover, free radicals activate inducible nitric oxide synthase (iNOS) which causes excessive NO production. Intensified expression of iNOS has been detected virtually in all cell types tested including macrophages, fibroblasts, chondrocytes, osteoclasts, and epithelial cells and results in the production of large amounts of NO in animals and patients with inflammatory diseases.10,11 It has been demonstrated that SWT causes rapid enhancement of endothelial nitric oxide synthase (eNOS) activity in SWT-treated cells resulting in increased NO levels.3 Moreover, it was reported that shock waves stimulate the NO-cyclic 30 -50 -guanosine monophosphate signaling pathway and increase mean plasma nitrite levels in patient with renal stones.12 However, the relationship between NO production and SWT-induced renal injury is still not understood. N-[3(Aminomethyl) benzyl) acetamidine] (1400W) is a highly selective iNOS inhibitor, which decreases NO production following ischemia reperfusion process in both kidney and heart in experimental studies.13,14 On the basis of these considerations, we aimed to evaluate the effect of inhibition of NO production by 1400W at SWT-induced kidney damage.
Materials and methods Animals The project was approved by the Experimental Ethics Committee of Gulhane Military Medical Academy, Ankara, Turkey and the National Institute of Health’s Guide for the Care and Use of Laboratory Animals was followed. Twenty-four male Sprague-Dawley rats, weighing between 250 and 300 g, were provided by the Gulhane Military Medical Academy, Experimental Research Committee, and housed in standard cages at a constant temperature (24 C) and light–dark cycle in a controlled environment. Rats were under standard rat chow and water ad libitum. Following 12 h of fasting period, animals were anesthetized with an intra-peritoneal injection of ketamine hydrochloride (50 mg/kg) and xylazine (10 mg/kg). The rats were placed on a heating pad and kept at 39 C to maintain constant body temperature. With an anterior midline abdominal incision, right kidney was removed, and clips were placed in the left renal fascia as the X-ray markers. After nephrectomy, in order to obtain a metabolic equilibration, rats were kept in their cages for 10 days. Rats were randomly divided into three groups; (1) control group (n ¼ 8), (2) SWT group (n ¼ 8), (3) SWT + 1400W group (n ¼ 8). 1400W (W4262, Sigma Chemical, St Louis, MO) was administered at a dose of 10 mg/kg at 2 h prior to SWT procedure and at the beginning of SWT procedure via intraperitoneal route and continued daily administration for consecutive 3 days. SWT procedure Under anesthesia, rats in the SWT and SWT + 1400W groups were fixed in supine position on the platform of lithotriptor and applied with shock wave lithotripsy at the left kidney under the guidance of X-rays. Each rat received 2000 shocks,
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18 kV, and total 15 J energy delivered with a Siemens LithoskopÔ (Munich, Germany). The focal size is 16 mm, the pressure range is not known according to technical brochure and the focal depth is 16 cm. The shock wave rate was 60 shock waves/min. The shock waves were applied with ramping similar to human application. Applied the number of shock waves and the level of energy were decided based on previous studies and our preliminary studies.15,16 After SWT procedure, rats were returned to their cages. 1400W administration was continued daily for consecutive 3 days. After 24 h of last dose of agent, animals were killed by cervical dislocation. At the time of death, blood was collected by heart puncture for biochemical analyses and left kidneys were collected for histopathological evaluation and biochemical examination. Biochemical analyses Serum samples were used for the measurement of blood urea nitrogen (BUN) and serum creatinine (SCr) levels, which were used as indicators of impaired glomerular function, and aspartate aminotransferase (AST) and alkaline phosphatase (ALP), which was used as an indicator of renal injury.17 BUN, SCr, AST, and ALP determined with a spectrophotometric technique by the Olympus AU-2700 autoanalyzer using commercial kits (Olympus, Hamburg, Germany). The frozen tissues were homogenized in phosphate buffer (pH 7.4) by means of a homogenizator (Heidolph Diax 900; Heidolph Elektro GmbH, Kelhaim, Germany) on an ice cube. The supernatant was used for entire assay. Initially, the protein content of tissue homogenates was measured by the method of Lowry et al.18 with bovine serum albumin as the standard was used for all assays. The lipid peroxidation level was measured with the thiobarbituric acid (TBA) reaction by the method of Ohkawa et al.19 This method was used to obtain a spectrophotometric measurement (Helios, Epsilon; Thermo Spectronic, Madison, WI) of the color produced during the reaction to TBA with malondialdehyde (MDA) at 535 nm. The calculated MDA levels were expressed as mmol/g-protein. The superoxide dismutase (SOD) activity was assayed using the nitroblue tetrazolium (NBT) method of Sun et al. and modified by Durak et al.20 In this method, NBT was reduced to blue formazan by O 2 , which has a strong absorbance at 560 nm. One unit (U) of SOD is defined as the amount of protein that inhibits the rate of NBT reduction by 50%. The estimated SOD activity was expressed as units per gram protein. The glutathione peroxidase (GSH-Px) activity was measured using the method described by Paglia and Valentine21 in which GSH-Px activity was coupled with the oxidation of NADPH by glutathione reductase. The oxidation of NADPH was spectrophotometrically followed up at 340 nm at 37 C. The absorbance at 340 nm was recorded for 5 min. The activity was the slope of the lines as mmol of NADPH oxidized per minute. GSH-Px activity was presented as U/gprotein. The tissue nitrite–nitrate (NOx) level was measured using the method of Miranda et al.22 Serum samples were passed through 0.45-mm pore membrane nitrocellulose filters before
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NOx analysis. The NOx levels were detected by means of an ion chromatograph (Dionex ICS-1000, Sunnyvale, CA). Anion and guard columns (AS-9HC/AG-9HC, CS12A/ CG12A, Sunnyvale, CA) and automated suppression were used. NOx levels were quantified using separate standard solutions for each ion. Serum neopterin (NP) levels were determined by using a high pressure liquid chromatography (HPLC) system with a fluorescence detector as prescribed previously23,24 and presented as nmol/l.
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Histopathologic evaluation The halves of each animal’s kidney were taken for histopathologic evaluation. In all groups, samples of kidney were placed in 10% tamponed formalin and send to pathology for routine automatic tissue processing. Then they embedded into paraffin. All paraffin blocks were subsequently sectioned at 5 mm thickness and stained with hematoxylin and eosin. The sections were scored with a semiquantitative scale designed to evaluate the degree of renal damage. The kidneys were evaluated in terms of glomerular changes [congestion, hemorrhage, necrosis, intracapillary and extracapillary proliferation (crescent formation)], tubular changes (epithelial vacuolar degeneration, necrosis and regeneration) interstitial changes (edema, peritubular capillary congestion, hemorrhage and inflammation) and vascular changes (fibrinoid necrosis, and fibrointimal thickening). All section areas for each kidney slide were examined and assigned for severity of changes. The scoring system used was 0, normal; 1, focal and mild; 2, focal and severe; 3, diffuse and mild; 4, diffuse and severe. Total histopathologic injury score per kidney was calculated by addition of all scores. Blind analysis of the histological samples was performed by two independent experts. For demonstrating the iNOS expression, standard 5-mm sections were obtained and deparaffinized with xylene and rehydrated in graded alcohols. Then, the sections were immunostained with primary antibodies against iNOS (1:50, monoclonal, Enzo, EnzoLifesciences Ltd, Plymouth, PA) using the standard streptavidin–biotin peroxidase technique on an automatic device (Ventana, Benchmark XT, Ventana Medical Systems, Tucson, AZ). The expression of iNOS was evaluated qualitatively by a pathologist using a light microscope. Statistical analysis All analyses were performed with the Statistical Package for the Social Sciences (SPSS) statistical program (Software version 11.0, SPSS Inc., Chicago, IL). Differences among the groups were analyzed by the Kruskal–Wallis test. Dual comparisons among groups with significant values were evaluated with the Mann–Whitney U-test. p50.05 Value were considered significant.
Results Serum biochemical values All serum biochemical values are summarized in Table 1. Serum creatinine and BUN levels in the SWT and SWT + 1400W groups were significantly lower than those
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Table 1. Biochemical values in serum.
Groups
Control (n ¼ 8)
SWT (n ¼ 8)
SWT + 1400W (n ¼ 8)
Creatinine 0.63 (0.59–0.68) 0.42a (0.40–0.53) 0.48a (mg/dL) 44a BUN (mg/dL) 52 (39–58) 38a (32–48) AST (IU/L) 92 (70–102) 127a (96–181) 107a,b ALP (IU/L) 297 (239–387) 425a (387–515) 371a,b Neopterin 6.1 (4.6–6.4) 8.5a (6.1–11.3) 7.2a,b (nmol/L)
(0.39–0.57) (36–52) (87–143) (315–429) (6.5–9.6)
Notes: Data are expressed as median (Min–Max). p50.05 statistically different from control group. b p50.05 statistically different from SWT group. a
of the control group (p50.05). Although the values in SWT + 1400W group were higher than the values in the SWT group, the difference was not significant (p40.05). Serum AST and ALP levels were significantly higher in the SWT group than the other groups suggesting increased renal injury (p50.05). Renal injury markers (AST, ALP) were significantly lower in SWT + 1400W group than the SWT group (p50.05), but it was still higher than the control group (p50.05). Serum neopterin levels Serum NP level was significantly increased in the SWT group than the control group (p50.05). It was significantly decreased in the SWT + 1400W group when compared to the SWT group, but was still higher than the control group (p50.05, SWT + 1400W vs. SWT and control group) (Table 1). Tissue lipid peroxidation levels The MDA levels in the SWT group were significantly higher than the other groups indicating increased renal cellular damage (p50.05, SWT vs. the other groups). The MDA levels were decreased in the treatment group, but still higher than the control group (p50.05, SWT + 1400W group vs. the other groups) (Table 2). Tissue antioxidant enzyme activities The tissue SOD and GSH-Px activity were significantly increased in both groups subjected to SWT procedure (p50.05, SWT and SWT + 1400W groups vs. control group). Antioxidant enzyme activities were significantly decreased in the tissue of rats subjected to SWT procedure and administered 1400W when compared in those subjected to SWT procedure alone (p50.05, SWT + 1400W vs. SWT). However, antioxidant enzyme activities were still high in the SWT + 1400W group when compared to the control group (p50.05, SWT + 1400W groups vs. control group) (Table 2). Tissue NOx level (nitrite/nitrite concentration) There was a clear increase in the tissue NOx levels in the SWT group, suggesting increased NO and peroxynitrite production (p50.05, SWT group vs. control group). The NOx levels in the SWT + 1400 group were significantly lower than those in the SWT group (p50.05, SWT + 1400W group vs. SWT groups) (Table 2).
Effect of 1400W in SWT-induced renal injury
DOI: 10.3109/0886022X.2014.884396
Histological findings Microscopic examination of the kidneys revealed obvious mild to moderate glomerular congestion, and severe tubular injury, which are consisted of epithelial vacuolization and necrosis with necrotic luminal debris, diminishing or loss of brush borders, and moderate to severe interstitial edema, inflammation, and peritubular capillary congestion in the SWT group (Figure 1B) compared with the SWT + 1400W and control group (Figure 1C and A). The highest histopathological scores were in the SWT group. These scores were significantly different than the other groups (p50.001, Table 3). Tubular injury was severe (Figure 1B). In the SWT + 1400W group, glomerular, tubular, and interstitial
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changes were better than the SWT and control group (Figure 1C and A). Figure 2 shows the distribution of iNOS staining in the kidneys. In the control group, it is seen that iNOS is expressed mostly in the cortex, especially among the proximal tubular epithelium (Figure 2A). SWT resulted in intense staining of iNOS in the tubular structures and no staining in the glomerular structures (Figure 2B). This intensity was lower in the kidneys of SWT + 1400W group compared to the SWT group (Figure 2C).
Discussion Today, since SWT for the treatment of urinary system stones has gained a wide popularity, numerous clinical and
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Table 2. Values of lipid peroxidation and antioxidant enzymes. Groups MDA (nmol/g-pro) SOD (U/g-pro) GSH-Px (U/g-pro) NOx (mmol/g-tissue)
Control (n ¼ 8) 0.67 1696 25.4 78.5
(0.56–0.98) (539–812) (18.1–30.2) (64.5–87.6)
SWT (n ¼ 8) a
2.62 2791a 42.5a 95.5a
(1.85–3.27) (1908–3327) (37.5–52.4) (87.9–106.4)
SWT + 1400W (n ¼ 8) 1.84a,b 2137a,b 31.5a,b 85.1a,b
(1.66–2.34) (1917–2622) (26.3–38.1) (79.5–96.1)
Notes: Data are expressed as median (Min–Max). p50.05 statistically different from control group. b p50.05 statistically different from SWT group. a
Figure 1. Light microscopic examination of the kidneys. Figure A shows the kidney of control group. There was mild to moderate glomerular congestion, and severe tubular injury in SWT group (B). Tubular structures were preserved in the kidneys of SWT + 1400W groups (C) compared with the SWT group.
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Table 3. Pathologic scores.
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Groups Glomerular changes Congestion Hemorrhage Necrosis Crescent formation Tubular changes Vacuolar degeneration Necrosis Regeneration Interstitial changes Edema Congestion Hemorrhage Inflammation Vascular changes Fibrinoid changes Fibrointimal thickening Total score
Control (n ¼ 8)
SWT (n ¼ 8)
1 0 0 0
4 0 0 0
(0–1) (0–0) (0–0) (0–0)
(3–4)a (0–0) (0–0) (0–0)
SWT + 1400W (n ¼ 8) 3 0 0 0
(2–3)a (0–0) (0–0) (0–0)
1 (0–1) 0 (0–0) 0 (0–0)
4 (2–4)a 2 (1–2)a 2 (1–3)a
1 (0–2)b 0 (0–0)b 1 (0–2)b
0 1 0 0
1 3 0 1
0 3 0 0
(0–1) (0–1) (0–0) (0–0)
0 (0–0) 0 (0–0) 3 (0–4)
(0–1) (3–4)a (0–0) (0–2)
0 (0–0) 0 (0–0) 17 (9–21)a
Notes: Data are expressed as median (Min–Max). a p50.05 statistically different from control group. b p50.05 statistically different from SWT group.
(0–0) (2–4)a (0–0) (0–0)
0 (0–0) 0 (0–0) 8 (3–12)a,b
experimental studies have been reported presenting some evidence of side effects following SWT treatment. Renal injury is the most common side effect of this procedure. Concerning the mechanism of the side effects of SWT, although it was accepted that renal injury is resulted from the direct action of cavitations bubbles or shear stress originating from shock-wave energy, more recent studies showed that free radical formation originated from transient ischemia or impairment of renal hemodynamic due to SWT was considered to cause renal injury through indirect mechanism.4,25,26 Several clinical studies, measuring renal resistive index or followed renal blood velocity in the kidney subjected to SWT, showed that an increase in resistive index or a reduction in blood velocity indicative of vasoconstriction occurred.4,5,27 It has also been reported that there is a decrease in renal perfusion in kidney following SWT procedure by using dynamic gadolinium-DTPA enhanced magnetic resonance imaging.27 Studies focused on two possible explanations for the impairment of renal flow; renal sympathetic nerves may be activated by shock waves and vasoconstriction may be occurred by vasoconstrictors released from kidneys in response to the shock waves. As a result of this transient ischemic insult, it is thought that free oxygen radical
Figure 2. Immunostaining of the iNOS protein in the kidneys revealed intense positive staining (brown) in the tubular structure of kidneys subjected to SWT procedure (B). Immunostaining for iNOS was lower in the SWT+1400W group (C) when compared with the SWT group. Figure A shows the control group.
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DOI: 10.3109/0886022X.2014.884396
generation related to ischemia/reperfusion process occurs. This free oxygen radical generation is known to contribute parenchymal damage by lipid peroxidation and disruption of cellular membranes.26 The current study showed that SWT caused a considerable increase in the renal tissue level of MDA and antioxidant enzyme activities. It is well known that MDA serves as a reliable marker of free oxygen radical mediated lipid peroxidation.10,28 So, our results clearly showed that SWT procedure increases free oxygen radical causing renal cellular injury, especially that caused by peroxidation of cellular membrane lipids and oxidation of cellular proteins. However, we measured SOD and GSH-Px enzyme activities to evaluate renal oxidative stress level. Under physiological conditions, the harmful effects of superoxide are prevented by SOD, which converts superoxide to H2O2; then, GSH-Px converts H2O2 to water.29 However, during increased oxidative stress, these natural defenses may be activated by the excessive generation of ROS. Therefore, increased tissue antioxidant enzyme activities in the kidney subjected to SWT process in this study can be accepted as an indirect indicator of the excessive amounts of the free oxygen radical. It was reported that shock wave treatment causes an elevation in concentration of intracellular free radical in suspended cells in vitro.30 Recent clinical and experimental studies have also shown that energy released by SWT produces direct injury to the renal vasculature and/or renal parenchymal cells, which results in an increased free oxygen radical formation.5,8,9 Our results are consistent with these studies and strongly confirm that the kidney following SWT produces free oxygen radical and these radicals might be a possible reason of SWT induced kidney injury. Under physiologic conditions, NO maintains its vascular tone and inhibits aggregation and adhesion of neutrophils and platelets to vascular endothelium; these are beneficial aspects of NO function.31 Low levels of NO production protect an organ in the early stages of injury, whereas elevated and prolonged NO production by iNOS during the later stages of the insult result in or potentiate organ injury. It was also proposed that NO-induced cellular damage is attributable to a powerful and cytotoxic oxidant, ONOO, which is generated 29,32 by interaction of NO with O Production of ONOO 2 . (nitrosative stress) occurs almost instantaneously and causes the nitration of cellular proteins with subsequent damage to protein structure and function.6,29,31 Supporting these observations have come from various in vivo studies which shown that NO biosynthesis and its action are closely related to the pathogenesis of renal ischemia reperfusion injury.10,33 The role of NO in the development of renal ischemia reperfusion injury has been also confirmed in several investigations in which administration of iNOS inhibitors protected the kidney against ischemia reperfusion injury.31 In addition, it was shown that the kidneys of iNOS knockout mice are resistant to hypoxic injury, whereas eNOS knockout or neuronal NOS knockout mice were not.33 Taken into account these data, it is presumed that inhibition of NO production at an early stage of insult would prevent inflammation and subsequent tissue damage in kidney subjected to SWT procedure. Therefore, we administered 1400W to figure out
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whether NO formation causes damage in rats subjected to SWL procedure. 1400W is a known highly selective inhibitor for iNOS in rat tissue and administrated dose of agents in the current study was chosen by considering previous studies.34,35 Moreover, it is well known that release of mediators commence after acute inflammation and continue consecutive three days based on acute inflammation studies of animal models.36,37 To reach effective plasma concentration, 1400W (10 mg/kg body weight) was administered before SWT procedure and continued consecutive three days with the aim of defining the role of NO formation in acute phase of this process. The tissue NOx levels were evaluated to determine the affect of nitrosative stress. NO and ONOO are eventually converted to nitrite (NO2) and/or nitrate (NO3), that is, NOx, therefore NOx levels are used as an indirect but reliable indicator for NO and ONOO formation in vivo.38 We found that tissue NOx levels are significantly higher in kidneys subjected to SWT procedure. Moreover, administration of 1400W reduced tissue NOx levels in kidneys subjected to SWT procedure. This decrement showed a clear correlation with histopathologic injury scores. Immunohistochemistry has helped to identify the tubular and glomerular structures in which iNOS expression is augmented by SWT. In the kidneys of rats subjected to SWT procedure, extensive and prominent iNOS immunoreactivity was noted in the tubular structure, whereas iNOS immunostaining in the glomerular structure was none. These findings show correlation with light microscopic evaluations showing severe tubular injury in SWT group. A few reports support this observation. Park et al.12 reported that shock waves released NO from renal cells and they speculated that NO production may decrease renin production, preventing renal damage resulting from excessive renal vasoconstriction by the renin–angiotensin system. In addition, Sheng et al.9 reported that plasma NO levels increased in patients with nephrolithiasis after SWT. So, it can be concluded that renal injury following SWT originates from excessive production of NO and ONOO. Thus we can speculate that the medications inhibiting the expression of iNOS or scavenging of ONOO in the kidney might protect or recover the injured renal tissue against SWT. Neopterin is formed during the course of cell-mediated immune response and there is a strong correlation between detection of NP and ability of monocytes/macrophages to scavenge ROS.23,39 Therefore, increased NP determination in the SWT group is a marker for the amount of immunologically induced oxidative stress. As a conclusion, SWT is not a harm free treatment modality. As a side effect of SWT, renal injury is mostly localized at the proximal tubular system and this injury is due to excessive production of free oxygen radicals and NO production. Moreover, iNOS inhibitors or peroxynitrite scavengers might protect the kidneys against SWT-induced morphological and functional injury.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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References 1. Skolarikos A, Alivizatos G, de la Rosette J. Extracorporeal shock wave lithotripsy 25 years later: complications and their prevention. Eur Urol. 2006;50:981–990;discussion 990. 2. Sarica K, Yencilek F. Prevention of shockwave induced functional and morphological alterations: an overview. Arch Ital Urol Androl. 2008;80:27–33. 3. Mariotto S, de Prati AC, Cavalieri E, et al. Extracorporeal shock wave therapy in inflammatory diseases: molecular mechanism that triggers anti-inflammatory action. Curr Med Chem. 2009;16: 2366–2372. 4. Connors BA, Evan AP, Willis LR, et al. The effect of discharge voltage on renal injury and impairment caused by lithotripsy in the pig. J Am Soc Nephrol. 2000;11:310–318. 5. Yilmaz E, Mert C, Keskil Z, et al. Effect of SWL on renal hemodynamics: could a change in renal artery contraction-relaxation responses be the cause? Urol Res. 2012;40:775–780. 6. Korkmaz A, Manchester CL. Reactive nitrogen species; devastating intracellular players and melatonin as a defender. J Exp Integr Med. 2011;1:63–65. 7. Bas M, Tugcu V, Kemahli E, et al. Curcumin prevents shock-wave lithotripsy-induced renal injury through inhibition of nuclear factor kappa-B and inducible nitric oxide synthase activity in rats. Urol Res. 2009;37:159–164. 8. Serel TA, Ozguner F, Soyupek S. Prevention of shock waveinduced renal oxidative stress by melatonin: an experimental study. Urol Res. 2004;32:69–71. 9. Sheng B, He D, Zhao J, et al. The protective effects of the traditional Chinese herbs against renal damage induced by extracorporeal shock wave lithotripsy: a clinical study. Urol Res. 2011; 39:89–97. 10. Ersoz N, Guven A, Cayci T, et al. Comparison of the efficacy of melatonin and 1400W on renal ischemia/reperfusion injury: a role for inhibiting iNOS. Ren Fail. 2009;31:704–710. 11. Moncada S, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991;43: 109–142. 12. Park JK, Cui Y, Kim MK, et al. Effects of extracorporeal shock wave lithotripsy on plasma levels of nitric oxide and cyclic nucleotides in human subjects. J Urol. 2002;168:38–42. 13. Mark LA, Robinson AV, Schulak JA. Inhibition of nitric oxide synthase reduces renal ischemia/reperfusion injury. J Surg Res. 2005;129:236–241. 14. Ovechkin AV, Lominadze D, Sedoris KC, et al. Inhibition of inducible nitric oxide synthase attenuates platelet adhesion in subpleural arterioles caused by lung ischemia-reperfusion in rabbits. J Appl Physiol. 2005;99:2423–2432. 15. Carvalho M, Freitas Filho LG, Fagundes DJ, et al. Effects of repeated extracorporeal shock wave in urinary biochemical markers of rats. Acta Cir Bras. 2009;24:496–501. 16. Kira VM, Fagundes DJ, Bandeira CO, et al. Effects of repeated extracorporeal shock wave on kidney apoptosis of normal and diabetic rat. Int Braz J Urol. 2008;34:91–96. 17. Chatterjee PK, Patel NS, Kvale EO, et al. Inhibition of inducible nitric oxide synthase reduces renal ischemia/reperfusion injury. Kidney Int. 2002;61:862–871. 18. Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265–275. 19. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem. 1979;95: 351–358.
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20. Durak I, Yurtarslanl Z, Canbolat O, et al. A methodological approach to superoxide dismutase (SOD) activity assay based on inhibition of nitroblue tetrazolium (NBT) reduction. Clin Chim Acta. 1993;214:103–104. 21. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med. 1967;70:158–169. 22. Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide. 2001;5:62–71. 23. Cakir E, Akgul OE, Aydin I, et al. The association between neopterin and acetaminophen-induced nephrotoxicity. Ren Fail. 2010;32:740–746. 24. Agilli M, Yaman H, Cayci T, et al. Comparison of two different HPLC methods and elisa method for measurement of serum neopterin. J Invest Biochem. 2012;1:43–47. 25. Williams JC Jr, Stonehill MA, Colmenares K, et al. Effect of macroscopic air bubbles on cell lysis by shock wave lithotripsy in vitro. Ultrasound Med Biol. 1999;25:473–479. 26. Munver R, Delvecchio FC, Kuo RL, et al. In vivo assessment of free radical activity during shock wave lithotripsy using a microdialysis system: the renoprotective action of allopurinol. J Urol. 2002;167: 327–334. 27. Mostafavi MR, Chavez DR, Cannillo J, et al. Redistribution of renal blood flow after SWL evaluated by Gd-DTPA-enhanced magnetic resonance imaging. J Endourol. 1998;12:9–12. 28. Chen Z-H, Niki E. Two faces of lipid peroxidation products: the – Yin and Yang – principles of oxidative stress. J Exp Integr Med. 2011;1:215–219. 29. Guven A, Uysal B, Akgul O, et al. Scavenging of peroxynitrite reduces renal ischemia/reperfusion injury. Ren Fail. 2008;30: 747–754. 30. Suhr D, Brummer F, Hulser DF. Cavitation-generated free radicals during shock wave exposure: investigations with cell-free solutions and suspended cells. Ultrasound Med Biol. 1991;17:761–768. 31. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol. 1996; 271:C1424–C1437. 32. Chatterjee PK. Novel pharmacological approaches to the treatment of renal ischemia-reperfusion injury: a comprehensive review. Naunyn Schmiedebergs Arch Pharmacol. 2007;376:1–43. 33. Ling H, Gengaro PE, Edelstein CL, et al. Effect of hypoxia on proximal tubules isolated from nitric oxide synthase knockout mice. Kidney Int. 1998;53:1642–1646. 34. Garvey EP, Oplinger JA, Furfine ES, et al. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem. 1997;272:4959–4963. 35. Kesik V, Guven A, Vurucu S, et al. Melatonin and 1400W ameliorate both intestinal and remote organ injury following mesenteric ischemia/reperfusion. J Surg Res. 2009;157:e97–e105. 36. Lawrence T, Willoughby DA, Gilroy DW. Anti-inflammatory lipid mediators and insights into the resolution of inflammation. Nat Rev Immunol. 2002;2:787–795. 37. Guven A, Uysal B, Caliskan B, et al. Mercaptoethylguanidine attenuates caustic esophageal injury in rats: a role for scavenging of peroxynitrite. J Pediatr Surg. 2011;46:1746–1752. 38. Guven A, Gundogdu G, Uysal B, et al. Hyperbaric oxygen therapy reduces the severity of necrotizing enterocolitis in a neonatal rat model. J Pediatr Surg. 2009;44:534–540. 39. Akgul EO, Aydin I, Cayci T, et al. The indicator of cellular immune response in body fluids: neopterin. Gulhane Med J. 2013;55: 237–243.
LABORATORY STUDY
Inhibition of inducible nitric oxide synthase prevents shock wave therapy induced renal injury Bilal Firat Alp1, Ercan Malkoc2, Zafer Demirer1, Ali Guragac1, Turker Turker3, Ertan Altayli4, Ayhan Ozcan5, Bulent Uysal6, Turgut Topal6, Emin Ozgur Akgul7, Ibrahim Yildirim1, and Ahmet Guven8 1
Department of Urology, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, 2Division of Urology, Military Hospital, Tekirdag, Turkey, Department of Epidemiology, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, 4Department of Research and Development Center, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, 5Department of Pathology, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, 6Department of Physiology, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, 7Department of Biochemistry, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey, and 8Department of Pediatric Surgery, Gulhane Military Medical Faculty, Etlik, Ankara, Turkey
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Abstract
Keywords
Objectives: Shock wave lithotripsy treatment (SWT) is not completely free from side effects; one of the accused mechanisms for renal injury during SWT is oxygen- and nitrogen-derived free radical productions. Therefore, we aimed to evaluate the effect of inhibition of nitric oxide (NO) production by N-[3(aminomethyl) benzyl) acetamidine] (1400W), highly selective inducible nitric oxide synthase (iNOS) inhibitor, at SWT-induced kidney damage. Materials and methods: Twenty-four rats those underwent right nephrectomy procedure were divided equally into three groups as control, SWT, and SWT + 1400W. 1400W was administered at a dose of 10 mg/kg at 2 h prior to SWT procedure and at the beginning of SWT procedure via intraperitoneal route and continued daily for consecutive 3 days. At the end of the fourth day, animals were killed via decapitation and trunk blood and the left kidneys were taken for biochemical and histopathologic evaluation. Results: SWT caused renal tubular damage and increased lipid peroxidation and antioxidant enzyme activities and SWT also significantly increased nitro-oxidative products. Inhibition of iNOS via administration of 1400W ameliorated renal injury and decreased tissue lipid peroxidation (malondialdehyde), superoxide dismutase, glutathione peroxidase and nitrite/nitrate levels (NOx). In addition, it was seen that histolopathological changes were attenuated in the SWT + 1400W group when compared to SWT group. Conclusion: SWT-induced renal injury might be due to excessive production of oxygen free radicals and NO production. Inhibition of iNOS attenuates renal injury following SWT treatment. It can be concluded that iNOS inhibitors or peroxynitrite scavengers might be used to protect the kidneys against SWT-induced morphological and functional injuries.
1400W, iNOS inhibitor, oxidative stress, renal injury, shock wave therapy
Introduction Since its first presentation, extracorporeal shock wave lithotripsy treatment (SWT) has gained popularity in treating kidney stones because of its ease of use, noninvasive nature and high efficacy. However, despite its marked effectiveness, SWT is not completely free from side effects. The main target of the shockwaves applied during a SWT procedure are stones located in the kidney but the surrounding tissues, renal parenchyma, and other organs are traumatized during this procedure.1 For the last two decades, more reports evaluating the adverse effects of SWT with experimental as well as clinical
Address correspondence to Ahmet Guven, Department of Pediatric Surgery, Gulhane Military Medical Faculty, 06017 Etlik, Ankara, Turkey. Tel.: +90 312 3045484; Fax: +90 312 3042150; E-mail: [email protected]
History Received 10 September 2013 Revised 9 December 2013 Accepted 28 December 2013 Published online 10 February 2014
studies came out.2,3 Concerning the mechanism of the adverse effects of SWT, reports have suggested that, renal damage is induced by the physical force of the waves and transient ischemia caused by both local and regional vasoconstriction, and intra-parenchymal bleeding.4,5 As a result of these insults, release of cytokines/inflammatory cellular mediators, production of oxygen-derived [reactive oxygen species (ROS)] free radicals, such as superoxide (O 2 ), peroxide (H2O2) and hydroxyl radicals (OH), production of nitrogen-derived free radicals (reactive nitrogen species), such as nitric oxide (NO) peroxynitrite (ONOO), and infiltration of tissue by inflammatory response cells initiate the process of cellular damage and death.6 However, this hypothesis is supported by researches that a variety of antioxidant agents have been found to be effective in SWT induced renal parenchymal injury.7–9 Nevertheless, the biochemical mechanisms underlying tissue damage have yet to be fully elucidated. One of the mechanism discussed for tissue damage in SWT is free oxygen radical production.2 Hydroxyl radicals and
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DOI: 10.3109/0886022X.2014.884396
superoxide anions are well known oxygen free radicals, which impose cytotoxic effects on cells. Moreover, free radicals activate inducible nitric oxide synthase (iNOS) which causes excessive NO production. Intensified expression of iNOS has been detected virtually in all cell types tested including macrophages, fibroblasts, chondrocytes, osteoclasts, and epithelial cells and results in the production of large amounts of NO in animals and patients with inflammatory diseases.10,11 It has been demonstrated that SWT causes rapid enhancement of endothelial nitric oxide synthase (eNOS) activity in SWT-treated cells resulting in increased NO levels.3 Moreover, it was reported that shock waves stimulate the NO-cyclic 30 -50 -guanosine monophosphate signaling pathway and increase mean plasma nitrite levels in patient with renal stones.12 However, the relationship between NO production and SWT-induced renal injury is still not understood. N-[3(Aminomethyl) benzyl) acetamidine] (1400W) is a highly selective iNOS inhibitor, which decreases NO production following ischemia reperfusion process in both kidney and heart in experimental studies.13,14 On the basis of these considerations, we aimed to evaluate the effect of inhibition of NO production by 1400W at SWT-induced kidney damage.
Materials and methods Animals The project was approved by the Experimental Ethics Committee of Gulhane Military Medical Academy, Ankara, Turkey and the National Institute of Health’s Guide for the Care and Use of Laboratory Animals was followed. Twenty-four male Sprague-Dawley rats, weighing between 250 and 300 g, were provided by the Gulhane Military Medical Academy, Experimental Research Committee, and housed in standard cages at a constant temperature (24 C) and light–dark cycle in a controlled environment. Rats were under standard rat chow and water ad libitum. Following 12 h of fasting period, animals were anesthetized with an intra-peritoneal injection of ketamine hydrochloride (50 mg/kg) and xylazine (10 mg/kg). The rats were placed on a heating pad and kept at 39 C to maintain constant body temperature. With an anterior midline abdominal incision, right kidney was removed, and clips were placed in the left renal fascia as the X-ray markers. After nephrectomy, in order to obtain a metabolic equilibration, rats were kept in their cages for 10 days. Rats were randomly divided into three groups; (1) control group (n ¼ 8), (2) SWT group (n ¼ 8), (3) SWT + 1400W group (n ¼ 8). 1400W (W4262, Sigma Chemical, St Louis, MO) was administered at a dose of 10 mg/kg at 2 h prior to SWT procedure and at the beginning of SWT procedure via intraperitoneal route and continued daily administration for consecutive 3 days. SWT procedure Under anesthesia, rats in the SWT and SWT + 1400W groups were fixed in supine position on the platform of lithotriptor and applied with shock wave lithotripsy at the left kidney under the guidance of X-rays. Each rat received 2000 shocks,
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18 kV, and total 15 J energy delivered with a Siemens LithoskopÔ (Munich, Germany). The focal size is 16 mm, the pressure range is not known according to technical brochure and the focal depth is 16 cm. The shock wave rate was 60 shock waves/min. The shock waves were applied with ramping similar to human application. Applied the number of shock waves and the level of energy were decided based on previous studies and our preliminary studies.15,16 After SWT procedure, rats were returned to their cages. 1400W administration was continued daily for consecutive 3 days. After 24 h of last dose of agent, animals were killed by cervical dislocation. At the time of death, blood was collected by heart puncture for biochemical analyses and left kidneys were collected for histopathological evaluation and biochemical examination. Biochemical analyses Serum samples were used for the measurement of blood urea nitrogen (BUN) and serum creatinine (SCr) levels, which were used as indicators of impaired glomerular function, and aspartate aminotransferase (AST) and alkaline phosphatase (ALP), which was used as an indicator of renal injury.17 BUN, SCr, AST, and ALP determined with a spectrophotometric technique by the Olympus AU-2700 autoanalyzer using commercial kits (Olympus, Hamburg, Germany). The frozen tissues were homogenized in phosphate buffer (pH 7.4) by means of a homogenizator (Heidolph Diax 900; Heidolph Elektro GmbH, Kelhaim, Germany) on an ice cube. The supernatant was used for entire assay. Initially, the protein content of tissue homogenates was measured by the method of Lowry et al.18 with bovine serum albumin as the standard was used for all assays. The lipid peroxidation level was measured with the thiobarbituric acid (TBA) reaction by the method of Ohkawa et al.19 This method was used to obtain a spectrophotometric measurement (Helios, Epsilon; Thermo Spectronic, Madison, WI) of the color produced during the reaction to TBA with malondialdehyde (MDA) at 535 nm. The calculated MDA levels were expressed as mmol/g-protein. The superoxide dismutase (SOD) activity was assayed using the nitroblue tetrazolium (NBT) method of Sun et al. and modified by Durak et al.20 In this method, NBT was reduced to blue formazan by O 2 , which has a strong absorbance at 560 nm. One unit (U) of SOD is defined as the amount of protein that inhibits the rate of NBT reduction by 50%. The estimated SOD activity was expressed as units per gram protein. The glutathione peroxidase (GSH-Px) activity was measured using the method described by Paglia and Valentine21 in which GSH-Px activity was coupled with the oxidation of NADPH by glutathione reductase. The oxidation of NADPH was spectrophotometrically followed up at 340 nm at 37 C. The absorbance at 340 nm was recorded for 5 min. The activity was the slope of the lines as mmol of NADPH oxidized per minute. GSH-Px activity was presented as U/gprotein. The tissue nitrite–nitrate (NOx) level was measured using the method of Miranda et al.22 Serum samples were passed through 0.45-mm pore membrane nitrocellulose filters before
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NOx analysis. The NOx levels were detected by means of an ion chromatograph (Dionex ICS-1000, Sunnyvale, CA). Anion and guard columns (AS-9HC/AG-9HC, CS12A/ CG12A, Sunnyvale, CA) and automated suppression were used. NOx levels were quantified using separate standard solutions for each ion. Serum neopterin (NP) levels were determined by using a high pressure liquid chromatography (HPLC) system with a fluorescence detector as prescribed previously23,24 and presented as nmol/l.
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Histopathologic evaluation The halves of each animal’s kidney were taken for histopathologic evaluation. In all groups, samples of kidney were placed in 10% tamponed formalin and send to pathology for routine automatic tissue processing. Then they embedded into paraffin. All paraffin blocks were subsequently sectioned at 5 mm thickness and stained with hematoxylin and eosin. The sections were scored with a semiquantitative scale designed to evaluate the degree of renal damage. The kidneys were evaluated in terms of glomerular changes [congestion, hemorrhage, necrosis, intracapillary and extracapillary proliferation (crescent formation)], tubular changes (epithelial vacuolar degeneration, necrosis and regeneration) interstitial changes (edema, peritubular capillary congestion, hemorrhage and inflammation) and vascular changes (fibrinoid necrosis, and fibrointimal thickening). All section areas for each kidney slide were examined and assigned for severity of changes. The scoring system used was 0, normal; 1, focal and mild; 2, focal and severe; 3, diffuse and mild; 4, diffuse and severe. Total histopathologic injury score per kidney was calculated by addition of all scores. Blind analysis of the histological samples was performed by two independent experts. For demonstrating the iNOS expression, standard 5-mm sections were obtained and deparaffinized with xylene and rehydrated in graded alcohols. Then, the sections were immunostained with primary antibodies against iNOS (1:50, monoclonal, Enzo, EnzoLifesciences Ltd, Plymouth, PA) using the standard streptavidin–biotin peroxidase technique on an automatic device (Ventana, Benchmark XT, Ventana Medical Systems, Tucson, AZ). The expression of iNOS was evaluated qualitatively by a pathologist using a light microscope. Statistical analysis All analyses were performed with the Statistical Package for the Social Sciences (SPSS) statistical program (Software version 11.0, SPSS Inc., Chicago, IL). Differences among the groups were analyzed by the Kruskal–Wallis test. Dual comparisons among groups with significant values were evaluated with the Mann–Whitney U-test. p50.05 Value were considered significant.
Results Serum biochemical values All serum biochemical values are summarized in Table 1. Serum creatinine and BUN levels in the SWT and SWT + 1400W groups were significantly lower than those
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Table 1. Biochemical values in serum.
Groups
Control (n ¼ 8)
SWT (n ¼ 8)
SWT + 1400W (n ¼ 8)
Creatinine 0.63 (0.59–0.68) 0.42a (0.40–0.53) 0.48a (mg/dL) 44a BUN (mg/dL) 52 (39–58) 38a (32–48) AST (IU/L) 92 (70–102) 127a (96–181) 107a,b ALP (IU/L) 297 (239–387) 425a (387–515) 371a,b Neopterin 6.1 (4.6–6.4) 8.5a (6.1–11.3) 7.2a,b (nmol/L)
(0.39–0.57) (36–52) (87–143) (315–429) (6.5–9.6)
Notes: Data are expressed as median (Min–Max). p50.05 statistically different from control group. b p50.05 statistically different from SWT group. a
of the control group (p50.05). Although the values in SWT + 1400W group were higher than the values in the SWT group, the difference was not significant (p40.05). Serum AST and ALP levels were significantly higher in the SWT group than the other groups suggesting increased renal injury (p50.05). Renal injury markers (AST, ALP) were significantly lower in SWT + 1400W group than the SWT group (p50.05), but it was still higher than the control group (p50.05). Serum neopterin levels Serum NP level was significantly increased in the SWT group than the control group (p50.05). It was significantly decreased in the SWT + 1400W group when compared to the SWT group, but was still higher than the control group (p50.05, SWT + 1400W vs. SWT and control group) (Table 1). Tissue lipid peroxidation levels The MDA levels in the SWT group were significantly higher than the other groups indicating increased renal cellular damage (p50.05, SWT vs. the other groups). The MDA levels were decreased in the treatment group, but still higher than the control group (p50.05, SWT + 1400W group vs. the other groups) (Table 2). Tissue antioxidant enzyme activities The tissue SOD and GSH-Px activity were significantly increased in both groups subjected to SWT procedure (p50.05, SWT and SWT + 1400W groups vs. control group). Antioxidant enzyme activities were significantly decreased in the tissue of rats subjected to SWT procedure and administered 1400W when compared in those subjected to SWT procedure alone (p50.05, SWT + 1400W vs. SWT). However, antioxidant enzyme activities were still high in the SWT + 1400W group when compared to the control group (p50.05, SWT + 1400W groups vs. control group) (Table 2). Tissue NOx level (nitrite/nitrite concentration) There was a clear increase in the tissue NOx levels in the SWT group, suggesting increased NO and peroxynitrite production (p50.05, SWT group vs. control group). The NOx levels in the SWT + 1400 group were significantly lower than those in the SWT group (p50.05, SWT + 1400W group vs. SWT groups) (Table 2).
Effect of 1400W in SWT-induced renal injury
DOI: 10.3109/0886022X.2014.884396
Histological findings Microscopic examination of the kidneys revealed obvious mild to moderate glomerular congestion, and severe tubular injury, which are consisted of epithelial vacuolization and necrosis with necrotic luminal debris, diminishing or loss of brush borders, and moderate to severe interstitial edema, inflammation, and peritubular capillary congestion in the SWT group (Figure 1B) compared with the SWT + 1400W and control group (Figure 1C and A). The highest histopathological scores were in the SWT group. These scores were significantly different than the other groups (p50.001, Table 3). Tubular injury was severe (Figure 1B). In the SWT + 1400W group, glomerular, tubular, and interstitial
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changes were better than the SWT and control group (Figure 1C and A). Figure 2 shows the distribution of iNOS staining in the kidneys. In the control group, it is seen that iNOS is expressed mostly in the cortex, especially among the proximal tubular epithelium (Figure 2A). SWT resulted in intense staining of iNOS in the tubular structures and no staining in the glomerular structures (Figure 2B). This intensity was lower in the kidneys of SWT + 1400W group compared to the SWT group (Figure 2C).
Discussion Today, since SWT for the treatment of urinary system stones has gained a wide popularity, numerous clinical and
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Table 2. Values of lipid peroxidation and antioxidant enzymes. Groups MDA (nmol/g-pro) SOD (U/g-pro) GSH-Px (U/g-pro) NOx (mmol/g-tissue)
Control (n ¼ 8) 0.67 1696 25.4 78.5
(0.56–0.98) (539–812) (18.1–30.2) (64.5–87.6)
SWT (n ¼ 8) a
2.62 2791a 42.5a 95.5a
(1.85–3.27) (1908–3327) (37.5–52.4) (87.9–106.4)
SWT + 1400W (n ¼ 8) 1.84a,b 2137a,b 31.5a,b 85.1a,b
(1.66–2.34) (1917–2622) (26.3–38.1) (79.5–96.1)
Notes: Data are expressed as median (Min–Max). p50.05 statistically different from control group. b p50.05 statistically different from SWT group. a
Figure 1. Light microscopic examination of the kidneys. Figure A shows the kidney of control group. There was mild to moderate glomerular congestion, and severe tubular injury in SWT group (B). Tubular structures were preserved in the kidneys of SWT + 1400W groups (C) compared with the SWT group.
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Table 3. Pathologic scores.
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Groups Glomerular changes Congestion Hemorrhage Necrosis Crescent formation Tubular changes Vacuolar degeneration Necrosis Regeneration Interstitial changes Edema Congestion Hemorrhage Inflammation Vascular changes Fibrinoid changes Fibrointimal thickening Total score
Control (n ¼ 8)
SWT (n ¼ 8)
1 0 0 0
4 0 0 0
(0–1) (0–0) (0–0) (0–0)
(3–4)a (0–0) (0–0) (0–0)
SWT + 1400W (n ¼ 8) 3 0 0 0
(2–3)a (0–0) (0–0) (0–0)
1 (0–1) 0 (0–0) 0 (0–0)
4 (2–4)a 2 (1–2)a 2 (1–3)a
1 (0–2)b 0 (0–0)b 1 (0–2)b
0 1 0 0
1 3 0 1
0 3 0 0
(0–1) (0–1) (0–0) (0–0)
0 (0–0) 0 (0–0) 3 (0–4)
(0–1) (3–4)a (0–0) (0–2)
0 (0–0) 0 (0–0) 17 (9–21)a
Notes: Data are expressed as median (Min–Max). a p50.05 statistically different from control group. b p50.05 statistically different from SWT group.
(0–0) (2–4)a (0–0) (0–0)
0 (0–0) 0 (0–0) 8 (3–12)a,b
experimental studies have been reported presenting some evidence of side effects following SWT treatment. Renal injury is the most common side effect of this procedure. Concerning the mechanism of the side effects of SWT, although it was accepted that renal injury is resulted from the direct action of cavitations bubbles or shear stress originating from shock-wave energy, more recent studies showed that free radical formation originated from transient ischemia or impairment of renal hemodynamic due to SWT was considered to cause renal injury through indirect mechanism.4,25,26 Several clinical studies, measuring renal resistive index or followed renal blood velocity in the kidney subjected to SWT, showed that an increase in resistive index or a reduction in blood velocity indicative of vasoconstriction occurred.4,5,27 It has also been reported that there is a decrease in renal perfusion in kidney following SWT procedure by using dynamic gadolinium-DTPA enhanced magnetic resonance imaging.27 Studies focused on two possible explanations for the impairment of renal flow; renal sympathetic nerves may be activated by shock waves and vasoconstriction may be occurred by vasoconstrictors released from kidneys in response to the shock waves. As a result of this transient ischemic insult, it is thought that free oxygen radical
Figure 2. Immunostaining of the iNOS protein in the kidneys revealed intense positive staining (brown) in the tubular structure of kidneys subjected to SWT procedure (B). Immunostaining for iNOS was lower in the SWT+1400W group (C) when compared with the SWT group. Figure A shows the control group.
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DOI: 10.3109/0886022X.2014.884396
generation related to ischemia/reperfusion process occurs. This free oxygen radical generation is known to contribute parenchymal damage by lipid peroxidation and disruption of cellular membranes.26 The current study showed that SWT caused a considerable increase in the renal tissue level of MDA and antioxidant enzyme activities. It is well known that MDA serves as a reliable marker of free oxygen radical mediated lipid peroxidation.10,28 So, our results clearly showed that SWT procedure increases free oxygen radical causing renal cellular injury, especially that caused by peroxidation of cellular membrane lipids and oxidation of cellular proteins. However, we measured SOD and GSH-Px enzyme activities to evaluate renal oxidative stress level. Under physiological conditions, the harmful effects of superoxide are prevented by SOD, which converts superoxide to H2O2; then, GSH-Px converts H2O2 to water.29 However, during increased oxidative stress, these natural defenses may be activated by the excessive generation of ROS. Therefore, increased tissue antioxidant enzyme activities in the kidney subjected to SWT process in this study can be accepted as an indirect indicator of the excessive amounts of the free oxygen radical. It was reported that shock wave treatment causes an elevation in concentration of intracellular free radical in suspended cells in vitro.30 Recent clinical and experimental studies have also shown that energy released by SWT produces direct injury to the renal vasculature and/or renal parenchymal cells, which results in an increased free oxygen radical formation.5,8,9 Our results are consistent with these studies and strongly confirm that the kidney following SWT produces free oxygen radical and these radicals might be a possible reason of SWT induced kidney injury. Under physiologic conditions, NO maintains its vascular tone and inhibits aggregation and adhesion of neutrophils and platelets to vascular endothelium; these are beneficial aspects of NO function.31 Low levels of NO production protect an organ in the early stages of injury, whereas elevated and prolonged NO production by iNOS during the later stages of the insult result in or potentiate organ injury. It was also proposed that NO-induced cellular damage is attributable to a powerful and cytotoxic oxidant, ONOO, which is generated 29,32 by interaction of NO with O Production of ONOO 2 . (nitrosative stress) occurs almost instantaneously and causes the nitration of cellular proteins with subsequent damage to protein structure and function.6,29,31 Supporting these observations have come from various in vivo studies which shown that NO biosynthesis and its action are closely related to the pathogenesis of renal ischemia reperfusion injury.10,33 The role of NO in the development of renal ischemia reperfusion injury has been also confirmed in several investigations in which administration of iNOS inhibitors protected the kidney against ischemia reperfusion injury.31 In addition, it was shown that the kidneys of iNOS knockout mice are resistant to hypoxic injury, whereas eNOS knockout or neuronal NOS knockout mice were not.33 Taken into account these data, it is presumed that inhibition of NO production at an early stage of insult would prevent inflammation and subsequent tissue damage in kidney subjected to SWT procedure. Therefore, we administered 1400W to figure out
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whether NO formation causes damage in rats subjected to SWL procedure. 1400W is a known highly selective inhibitor for iNOS in rat tissue and administrated dose of agents in the current study was chosen by considering previous studies.34,35 Moreover, it is well known that release of mediators commence after acute inflammation and continue consecutive three days based on acute inflammation studies of animal models.36,37 To reach effective plasma concentration, 1400W (10 mg/kg body weight) was administered before SWT procedure and continued consecutive three days with the aim of defining the role of NO formation in acute phase of this process. The tissue NOx levels were evaluated to determine the affect of nitrosative stress. NO and ONOO are eventually converted to nitrite (NO2) and/or nitrate (NO3), that is, NOx, therefore NOx levels are used as an indirect but reliable indicator for NO and ONOO formation in vivo.38 We found that tissue NOx levels are significantly higher in kidneys subjected to SWT procedure. Moreover, administration of 1400W reduced tissue NOx levels in kidneys subjected to SWT procedure. This decrement showed a clear correlation with histopathologic injury scores. Immunohistochemistry has helped to identify the tubular and glomerular structures in which iNOS expression is augmented by SWT. In the kidneys of rats subjected to SWT procedure, extensive and prominent iNOS immunoreactivity was noted in the tubular structure, whereas iNOS immunostaining in the glomerular structure was none. These findings show correlation with light microscopic evaluations showing severe tubular injury in SWT group. A few reports support this observation. Park et al.12 reported that shock waves released NO from renal cells and they speculated that NO production may decrease renin production, preventing renal damage resulting from excessive renal vasoconstriction by the renin–angiotensin system. In addition, Sheng et al.9 reported that plasma NO levels increased in patients with nephrolithiasis after SWT. So, it can be concluded that renal injury following SWT originates from excessive production of NO and ONOO. Thus we can speculate that the medications inhibiting the expression of iNOS or scavenging of ONOO in the kidney might protect or recover the injured renal tissue against SWT. Neopterin is formed during the course of cell-mediated immune response and there is a strong correlation between detection of NP and ability of monocytes/macrophages to scavenge ROS.23,39 Therefore, increased NP determination in the SWT group is a marker for the amount of immunologically induced oxidative stress. As a conclusion, SWT is not a harm free treatment modality. As a side effect of SWT, renal injury is mostly localized at the proximal tubular system and this injury is due to excessive production of free oxygen radicals and NO production. Moreover, iNOS inhibitors or peroxynitrite scavengers might protect the kidneys against SWT-induced morphological and functional injury.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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