Stesura Seveso 335Archivio Italiano di Urologia e Andrologia 2020; 92, 4 ORIGINAL PAPER No conflict of interest declared. DOI: 10.4081/aiua.2020.4.335 Effects of butein on renal ischemia/reperfusion injury: An experimental study Mehmet Akif Ramazanoglu 1, Tuncay Toprak 2, Mehmet Remzi Erdem 3, Gulistan Gumrukcu 4, Hatice Kucuk 5, Feridun Sengor 6 1 Department of Urology, Rize State Hospital, Rize, Turkey; 2 University of Health Sciences, Turkey. Fatih Sultan Mehmet Training and Research Hospital, Department of Urology, Istanbul, Turkey; 3 Department of Urology, Istanbul Kolan Hospital, Istanbul, Turkey; 4 Department of Pathology, Haydarpaşa Numune Training and Research Hospital, Istanbul, Turkey; 5 Department of Pathology, Kanuni Training and Research Hospital, Trabzon, Turkey; 6 Department of Pathology, University of Kırklareli, Faculty of Kırklareli, Turkey. mation, reactive oxygen radicals, apoptosis and necrosis (3, 4). Tissues that have undergone I/R are subjected to pro-inflammatory processes of cytokine release and the production of reactive oxygen radicals (ROR) by neu- trophils (5). The production of ROR is considered a key reason for uncontrolled oxidative stress during the reperfusion period (4). Thus, oxidative stress is extreme- ly important in renal I/R injury (6), and targeting its processes is an ideal therapeutic approach. The importance of I/R injury preventing during nephron sparing surgery is major issue in urology practice. Serum creatinine can increase and glomerular infiltration rate can decrease after nephron sparing surgery even in the patients who have healthy contralateral kidney. To pre- serve better kidney function leads lowering the risk of development of metabolic or cardiovascular disorders (7). To date, many pharmacological agents such as N- acetylcysteine (8), Allopurinal (9) or Mannitol (10) have been identified to decrease I/R injury after nephron spar- ing surgery. Another potential compound that can target I/R injury is butein, a polyphenolic compound which has reported biological activities ranging from antioxi- dant properties (11) to anti-fibrogenic, anti-inflamatory (12). It can also exert a protective effect on ischemia or I/R damage (13). Against this background, we hypothesized that butein using may reduce I/R injury in rats by its anti-oxidative effects. To the best of our knowledge, there is no infor- mation about of the anti-oxidative effects of butein in rat model of renal I/R injury. MATERIALS AND METHODS All experimental and surgical procedures were approved by the Institutional Animal Care and Use Committee of University of Bezmialem (Istanbul, Turkey) (Approval Number/ID: 2016/134). The procedures complied with the Guide for the Care and Use of Laboratory Animals and were conducted according to animal care guidelines (14). A total of 27 male Wistar- Albino (WA) rats (8 weeks old, weight 220-250 g) were purchased from the University of Bezmialem (Istanbul, Objectives: Renal ischemia/reperfusion (I/R) injury is a common cause of acute kidney injury. The aim of this study was to investigate the effect of butein on renal I/R injury. Materials and methods: Twenty-seven rats were randomly allocated to three groups (n = 9): a sham group, a renal I/R- untreated (control) group, and a renal I/R-butein group. The sham group underwent only opening and closing of the peritoneum. In the control group, an experimental I/R model was created and 1 cc isotonic saline was applied to the peri- toneum. In the butein group, the experimental I/R model was created and 1 mg/kg butein was administered intraperitoneally 15 minutes before the beginning of ischemia. The left kidneys of the rats were histopathologically examined for tissue dam- age caused by I/R. Results: Histopathological examination of the tissue damage revealed that all kidneys in the sham group were normal. By contrast, 2 in the control group (22.2%) had small focal dam- aged areas, 1 (11.1%) had < 10% cortical damage, 5 (55.6%) had 10-25% cortical damage, and 1 (11.1%) had 25-75% corti- cal damage. The butein group had 1 (11.1%) normal kidney, 2 (22.2%) with small focal damaged areas, 4 (44.4%) with < 10% cortical damage, and 2 (22.2%) with 10-25% cortical damage. Tissue damage was significantly lower in the sham group than in the control and butein groups (p < 0.01). No statistically significant differences were observed in the histopathology of the control and butein groups (p > 0.05). Conclusions: Intraperitoneal administration of butein had no significant effect on renal tissue injury. KEY WORDS: Butein; Oxidative stress; Renal ischemia; Reperfusion injury; Rat. Submitted 19 June 2020; Accepted 28 July 2020 INTRODUCTION Ischemia/reperfusion (I/R) injury is a common cause of renal injury arising from a variety of clinical circum- stances, including partial nephrectomy, renal transplan- tation, renal artery angioplasty, hydronephrosis and iatrogenic trauma (1, 2). The pathologic processes underlying this injury are complex and involve inflam- Summary Archivio Italiano di Urologia e Andrologia 2020; 92, 4 Mehmet Akif Ramazanoglu, Tuncay Toprak, Mehmet Remzi Erdem, Gulistan Gumrukcu, Hatice Kucuk, Feridun Sengor 336 Turkey). The animals were kept in captivity under the same nutritional and environmental conditions. Rats were entrained under a 12:12 h light: dark cycle with stable temperature (21 ± 2°C) and humidity (60 ± 10%). The rats had food and sterile water available ad libitum. Experimental design The WA rats were randomly divided into three groups (n = 9 in each group): 1. Sham group: After sterile conditions were obtained, a midline laparotomy was performed. The left kidney pedicle was then dissected. No other procedure was per- formed, and the incision was closed in two layers. 2. Control group (Renal I/R injury and plasebo group): After sterile conditions were obtained, a midline laparotomy was performed. Isotonic saline (1 mg/kg) was applied intraperitoneally 15 minutes before the beginning of arterial clamping. The left kidney pedicle was clamped with an artery clamp for 45 minutes. After 45 minutes of left renal ischemia, the occlusion clamp was removed and the incision was closed in two layers. 3. Renal I/R injury and butein group: After sterile condi- tions were obtained, a midline laparotomy was per- formed. Butein (1 mg/kg) was applied intraperitoneally 15 minutes before the beginning of arterial clamping. The left kidney pedicle was clamped with an artery clamp for 45 minutes. After 45 minutes of left renal ischemia, the occlusion clamp was removed and the inci- sion was closed in two layers. Butein was sourced from Farmasina Medical and Chemical Products Company (Kayışdağı, Ataşehir, Istanbul). Induction of renal I/R injury The rats were anesthetized with an intraperitoneal injec- tion of ketamine (100 mg/kg, Ketalar, Eczacıbasi, Turkey) and xylazine (10 mg/kg). The midline laparotomy and dissection of the left kidney pedicle were then performed as already described, followed by the I/R procedure for the control and butein groups. All procedures were per- formed under sterile conditions. The rats were sacrificed 24 h after completion of the reperfusion procedure and kidney samples were obtained. Histological analysis Renal tissues were extracted from rats in all groups (24 h after surgery), as well as following intraperitoneal injection of butein (1 mg/kg) in the butein group. Kidney tissue was embedded in paraffin and 5 μm tissue sections were taken for Hematoxylin and Eosin (H&E) staining. An independ- ent pathologist blinded to the treatment groups, analyzed three different tissue sections from rats in each treatment group, using a Zeiss Axio Imager A2 microscope (Carl Zeiss AG, Germany). The histological evaluations of the renal tissue were grad- ed as follows: 0, normal; 0.5, small focal damage areas; 1, areas of tubular epithelial cell necrosis and desquamation, including < 10% of cortical tubules; 2, similar changes, including 10-25% cortical tubules; 3, similar changes including 25-75% cortical tubules; and 4, similar changes including > 75% cortical tubules. This scoring system is shown in Table 1 (15). Statistical analysis Statistical analysis was performed using the SPSS 22 soft- ware. Before starting to study, we performed power analysis. The power analysis showed that 9 subjects per group would be needed to have a 80% chance of achiev- ing statistical significance at the p < 0.05 level. The Kolmogorov-Smirnov test was performed to deter- mine the normality of data. The results are expressed as mean ± SD. Mann-Whitney U and Kruskal-Wallis tests were performed for comparison of two and three inde- pendent samples, respectively. A p value below 0.05 was considered statistically significant. RESULTS Histopathological examination of tissue damage revealed that all rats in the sham group had normal kidneys. By contrast, 2 rats in the control group (22.2%) had small focal damaged areas, 1 (11.1%) had < 10% cortical dam- age, 5 (55.6%) had 10-25% cortical damage, and 1 (11.1%) had 25-75% cortical damage. The butein group had 1 rat (11.1%) with normal kidneys, 2 (22.2%) with small focal damaged areas, 4 (44.4%) with < 10% corti- cal damage, and 2 (22.2%) with 10-25% cortical dam- age. Renal cortical damage was significantly lower in the sham group than in the control and butein groups (p < 0.01). No statistically significant difference was noted in the histopathology of the control and butein groups (p > 0.05). Histopathologic results for the experimental animals were shown in Table 2. Histological images of the dam- age according to the scoring system for the rat renal cor- tex sections after IR injury were shown in Figures 1-4 Table 1. Scoring system for renal histopathology. Table 2. Histopathologic results for the experimental animals. Score Histopathological pattern 0 Normal 0.5 Small focal damaged areas 1 < 10% Cortical damaged zone 2 10–25% Cortical damaged zone 3 25–75% Cortical damaged zone 4 > 75% Cortical damaged zone Sham Control Butein Total group n (%) group n (%) group n (%) Normal 9 (100) 0 1 (11.1) 10 (37.0) Small focal damaged areas 0 2 (22.2) 2 (22.2) 4 (14.8) < 10% damaged cortical zone 0 1 (11.1) 4 (44.4) 5 (18.5) 10–25% damaged cortical zone 0 5 (55.6) 2 (22.2) 7 (25.9) 25–75% damaged cortical zone 0 1 (11.1) 0 1 (3.7) Min-max (median) 0–0 (0) 0.5-3 (2) 0-2 (1) 0-3 (0.5) pa 0.001** Sham-Control groupb 0.001** Sham-Butein groupb 0.001** Control-Butein groupb 0.105 a Kruskal-Wallis test; b Mann-Whitney U test; **p < 0.01. 337Archivio Italiano di Urologia e Andrologia 2020; 92, 4 Effects of butein in renal I/R injury and dissociation of histopathologic tissue damage according to groups was shown in Figure 5. DISCUSSION The complicated pathogenesis of renal I/R injury is due to the broad range of effects on vascular endothelial and tubular epithelial cells (16). These effects are character- ized by the cellular accumulation of waste products, imbalances in electrolytes and acid-base levels (17). Renal I/R injury induces an inflammatory response by triggering the immune systems. An infiltration of leuko- cytes then occurs in the site of inflammation, followed by activation of tubular epithelial cells (18, 19). The influx of neutrophils and macrophages into injured renal tissue results in the secretion of pro-inflammatory cytokines, including TNF-a, HMGB1, IL-6, and IL-1b (20), while the infiltrated cells themselves reduce blood flow in the kidney and disrupt microcirculation (21). Significant increases in pro-inflammatory cytokine levels have been reported following renal I/R injury in rats (22). In the present study, butein was examined for its poten- tial effects on regulating renal I/R injury. We considered that butein may serve a protective role in the rat model of renal I/R injury model by regulating the level of inflammatory cytokines. Butein is an important herbal polyphenol in traditional Japanese and Korean medicine, where it has been used as an analgesic, antibiotic, antithrombotic, anticancer, and anti-inflammatory med- icine (23). It has demonstrated a broad spectrum of activity in both in vivo and in vitro models, including antineoplastic, anti-inflammatory, and antioxidant effects (24), but the mechanism underlying its antiin- flammatory properties is unclear. The present evidence suggests that butein inhibits many enzymes and pro-inflammatory mediators and that it also suppresses macrophage-mediated inflammation. One study performed in macrophage culture showed suppression of lipopolysaccharide-dependent nitrite and PGE2 production by butein and a pronounced anti- inflammatory effect (25). Application of butein to cell cultures resulted in COX-1 and COX-2 inhibition (11). Lee and colleagues suggested the use of butein as a treatment for intestinal inflammation, as butein reduced the expression of IL-8 in intestinal epithelial cell cultures (26). Butein also reduced E-selectin expression in human umbili- cal vein cells and expression of ICAM-1 and VCAM-1 in endothelial cells (27). In addition, butein inhibited proinflam- matory gene activity by inhibiting NF- kBeta activation and inhibited the release of TNF-a, IL-6 and IL-8 (28). Many studies have shown that butein neutralizes and inhibits production of ROR and protects the cells from ROR damage. In living organisms, ROR arise as a result of normal biological metabo- lism and they can distort the structures of DNA, fats, proteins and carbohy- drates. The best known of these oxygen Figure 1. Normal cortex (Histopathologic score = 0). Figure 2. Cortical focal necrose areas (Histopathologic score = 1). Figure 3. More common cortical necrose areas (Histopathologic score = 2). Figure 4. Extensive cortical necrose areas (Histopathologic score = 3). Figure 5. Dissociation of histopathologic tissue damage according to groups. Archivio Italiano di Urologia e Andrologia 2020; 92, 4 Mehmet Akif Ramazanoglu, Tuncay Toprak, Mehmet Remzi Erdem, Gulistan Gumrukcu, Hatice Kucuk, Feridun Sengor 338 radicals are the superoxide anion, hydrogen peroxide, and hydroxyl radicals. Cheng et al. used CCl4 to cause cellular damage to the liver and then tried to repair this cellular damage using butein and a-tocopherol. Butein showed a similar antioxidant effect to that of a-toco- pherol, but butein had the advantage of eliciting an effect at lower doses (11). I/R-induced damage differs histologically in some respects from chemically induced damage. For example, the arterial circulation in the renal cortex is somewhat suppressed following I/R, due to tubulo-glomerular arte- riolar vasoconstriction, cellular swelling, tubular obstruc- tion, and vascular occlusion arising from extravasation of white blood cells and erythrocytes from the external medulla. The proximal tubules affected by I/R are dam- aged due to warm ischemia during recirculation and this leads to regression of the renal function when circulation returns to its original state. During re-infusion, the tubu- lar lumen diameter increases, while punctures from the proximal tubules block the tubules, creating resistance to fluid ingestion in the handle and proximal tubules. The result is a decrease in tubular reabsorption due to cel- lular damage, while capillary expansion increases the intratubular pressure in the external medullary collecting tubes. Subsequently, in addition to a 12% reduction in blood flow, the GFR is also reduced by 90% (29). The tubular tissue undergoes atrophy, the tubular lumen diameter increases, the tubules undergo hyalinization, and reactive atypia, brushy edge loss, cellular swelling, nucle- us deformity, and leukocyte infiltration are observed. In our study, histopathologic evaluation of the tissue response to butein in the renal I/R rat model was com- pared with the non-butein control group and the sham group. Histopathologically significant differences were found between the sham group and the other groups, but no statistically significance was noted histopatholog- ically between the I/R rats treated with butein or the saline control (p > 0.05). 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Correspondence Mehmet Akif Ramazanoglu, MD (Corresponding Author) maramazanoglu@hotmail.com Rize State Hospital, Department of Urology Eminettin mah. Atatürk caddesi, merkez, 53020 Rize (Turkey) Tuncay Toprak, MD drtuncay55@hotmail.com University of Health Sciences, Turkey. Fatih Sultan Mehmet Training and Research Hospital, Department of Urology, Istanbul (Turkey) Mehmet Remzi Erdem, MD remzierdem@gmail.com Department of Urology, Istanbul Kolan Hospital, Istanbul (Turkey) Gulistan Gumrukcu, MD Department of Pathology, Haydarpasa Numune Training and Research Hospital, Istanbul (Turkey) Hatice Kucuk, Assistant Professor dr.hatice.kucuk@hotmail.com Department of Pathology, Kanuni Training and Research Hospital, Trabzon (Turkey) Feridun Sengor, Professor fsengor2004@yahoo.com Department of Urology, University of Kırklareli, Faculty of Kırklareli, (Turkey)