Renal effects of 7,8-Dihydroxyflavone in cafeteria diet-induced obesity

Renal effects of 7,8-Dihydroxyflavone in cafeteria diet-induced obesity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Renal effects of 7,8-Dihydroxyflavone in cafeteria diet-induced obesity Emine Gulceri Gulec Peker, Selma Cirrik, Gulay Hacioglu, Elif Sahin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2053626/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective: In this study, the possible protective effect of 7,8-Dihydroxyflavone (7,8-DHF), a brain-derived neurotrophic factor (BDNF) mimetic and anti-oxidant flavonoid, in renal damage caused by cafeteria diet-induced obesity was investigated. Method: In the study, 4-5 week old C57BL/6 male mice were used and the subjects were divided into 4 groups as Control, CD (cafeteria diet), CD+Vehicle and CD+7,8-DHF (n=9-11). Control group subjects were fed with chow diet for 16 weeks and other groups were fed with cafeteria diet. In the last 28 days of the feeding period, 7,8-DHF treatment (5 mg/kg/day, intraperitoneal) was administered in the CD+7,8-DHF group, and DMSO (17%) as a 7,8-DHF carrier was administered in the CD+Vehicle group. At the end of 16 weeks, the subjects were sacrificed and malondialdehyde (MDA), reduced glutathione (GSH), nitrite + nitrate (NOx) and collagen levels, and superoxide dismutase (SOD) and catalase (CAT) enzyme activities were measured in kidney tissues. Results: At the end of 16 weeks, body weights of all subjects increased compared to baseline. Weight gain was higher in CD (p<0.001) and CD+Vehicle groups (p<0.001) compared to control. The weight gain in the CD+7,8-DHF group was not different from the control. Compared to the CD group, the weight gains in the CD+Vehicle and CD+7,8-DHF groups were lower. Compared to the control group CD group had higher renal MDA level (p<0.0001), lower GSH level (p<0.0001), less SOD (p<0.0001) and CAT (p<0.0001) activity, lower NOx (p<0.0001) and collagen (p<0.0001) levels. Vehicle administration did not affect these parameters as results were similar to CD group. However, significant changes were noted with 7,8-DHF treatment. Compared with the CD+Vehicle group, the CD+7,8-DHF group had lower MDA levels (p<0.001), higher GSH levels (p<0.001), lower NOx levels (p<0.001), higher SOD (p<0.001) and CAT (p<0.001) activities. 7,8-DHF treatment brought these parameters closer to the control values, but did not fully return to control, except for CAT activity. Renal collagen deposition was not affected by 7,8-DHF treatment. Conclusion: Oxidative stress plays an important role in obesity-induced renal damage. 7,8-DHF may be important in the suppression of renal damage in cafeteria diet-induced obesity, at least by inhibiting oxidative stress and excessive nitric oxide production. The increasing prevalence of eating habits and obesity together with the cafeteria diet in the society, makes these results clinically important. These effects of 7,8-DHF need to be investigated in more details. Cafeteria diet obesity-induced renal damage oxidative stress 7 8-DHF nitric oxide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Obesity is an important risk factor for chronic kidney disease as well as cardiovascular diseases and diabetes mellitus. Some changes that occur with obesity, for example, increase in plasma free fatty acids, hyperinsulinemia, insulin resistance, oxidative stress, proinflammatory conditions, decrease in adiponectin, leptin resistance, and activation of the renin-angiotensin-aldosterone system (RAAS) cause glomerular and tubular damage, inducing chronic predisposes for kidney disease (Pommer, 2018 ; Manna and Jain, 2015 , Wang et al., 2008 ). Hyperinsulinemia contributes to this process by generating endothelial dysfunction, increased oxidative stress, and pro-inflammatory and pro-fibrotic changes in the kidney. In addition, renal lipotoxicity, which occurs with the accumulation of triglycerides in the glomeruli and proximal tubules, is an important factor in kidney damage. Obesity-related glomerulopathy (ORG) is an important clinical manifestation of all these changes (Lakkis and Weir, 2018 ). Increased oxidative stress is accepted as one of the important mechanisms in the pathophysiology of obesity. The main factors that stimulate an increase in oxidative stress in obesity are hyperglycemia and the change in cell metabolism due to the increase in plasma free fatty acids and the increase in the production of reactive oxygen species (ROS) (Manna and Jain, 2015 , Sharma, 2016 ; Pellegrino et al., 2019 ). In addition, some proinflammatory cytokines such as TNF-α and IL-6 released from adipose tissue contribute to the increment in oxidative stress by activating NADPH oxidase (NOX). Another factor originating from adipose tissue that activates NOX is the hormone leptin, which is why there is hyperleptinemia in obese individuals (Manna and Jain, 2015 , Sharma, 2016 ; Pellegrino et al., 2019 ). These adipose tissue-derived factors (TNF-α, IL-6 and leptin) also cause the kidney to become more susceptible for the development of fibrosis (Declèves and Sharma, 2015 , Tang et al., 2012 ; Wolf and Ziyadeh, 2006 ). In addition to the increase in ROS production, the weakening of antioxidant activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) also contributes to the enlargement in oxidative damage in obesity (Ozata et al., 2002 ; Noeman et al., 2011 ). It has been shown that approaches to reduce oxidative stress largely prevent renal damage observed in obesity. For example, renal oxidative stress, glomerular-tubular damage and functional loss that occur in experimental obesity models induced by high-fat diet are significantly abolished in the presence of herbal extracts with known antioxidant effects such as Terminalia arjuna , Caralluma fimbriata , and Dendrobium moniliforme (Kanthe et al., 2021 ; Gujjala et al., 2016 ; Lee et al., 2012 ). 7,8-Dihydroxyflavone (7,8-DHF) is a natural flavonoid found in the leaves of Godmania aesculifolia , Tridax procumbens and primula tree. In vitro studies have shown that the molecule has radical scavenging effects and up-regulates antioxidant enzymes such as CAT, SOD and hemoxygenase-1 (HO-1) (Choi et al., 2017 ; Choi et al., 2016 ). It has even been shown in vitro that the production of proinflammatory mediators (eg, nitric oxide, prostaglandin E2, interleukin 1β) caused by lipopolysaccharide induction is inhibited in the presence of 7,8-DHF (Park et al., 2014 ). The most important feature of 7,8-DHF is that it can mimic the effects of brain-derived neurotrophic factor (BDNF), which is one of the major factors playing a role in neuron development, vitality and maintenance of normal cell functions. The molecule shows this effect by binding to the tyrosine receptor kinase B (TrkB), the main receptor of BDNF. Indeed, the attenuating effects of 7,8-DHF have been demonstrated in some diseases involving BDNF, such as Alzheimer's, Parkinson's, depression, and obesity (Emili et al., 2020 ). The BDNF/TrkB pathway plays an important role in the regulation of body weight. In rats, BDNF infusion into the lateral ventricle suppresses food intake and body weight gain (Pelleymounter et al., 1995 ). Ablation or mutation of this pathway results in hyperphagia and obesity (Gray et al., 2006 , Yeo et al., 2004 , Kernie et al. 2000 ). In addition, it has been presented that 7,8-DHF administration increases systemic energy consumption by stimulating uncoupling protein 1 (UCP1) expression and AMP-activated protein kinase (AMPK) activity in skeletal muscle, thus preventing the development of obesity. The lack of anti-obesity effect of 7,8-DHF in muscular TrkB-receptor knockout mice suggests that this effect is mediated by muscular TrkB receptors, not hypothalamus (Chan et al., 2015 ; Wood et al., 2018 ). As a result, it is clear that not only the central activation of the BDNF/TrkB pathway, but also its peripheral activation participates in the regulation of body weight. Among the obesity studies, in vivo studies displaying the effects of 7,8-DHF on the increase in oxidative stress are limited. In these studies, it was indicated that the increase in oxidative stress caused by high-fat diet in the hippocampus and liver was abolished by 7,8-DHF treatment in 4 weeks (Pandey et al., 2020 ; Kumar et al., 2019 ). Kidney tissue, on the other hand, has not been studied before in obesity, in terms of the effects of BDNF/TrkB pathway or 7,8-DHF. Based on previous studies showing that approaches to prevent the increase in oxidative stress reduce obesity-related renal damage, 7,8-DHF administration is also likely to have positive effects on the kidneys. Thus, in our study, we aimed to examine the renal effects of 7,8-DHF treatment in obese mice. In the experimental obesity model selected in this study, the mice were fed with both high-calorie and delicious human food, which is defined as the cafeteria diet, such as hazelnut biscuits, fish crackers, fruitcakes, potato chips and chocolate wafers. Since this type of nutrition and obesity is an increasingly common problem and obesity is an important risk factor for chronic kidney disease, we think it is important to reveal the effects of 7,8-DHF, which is known for its anti-obesity effects and antioxidant effects on the kidneys. 2. Material And Method Experimental animals and grouping In the study, 4–5 weeks old (17–20 g) male C57BL/6 mice were used. Experimental procedures were approved by the Local Ethics Committee for animal experiments ( Karadeniz Technical University Animal Experiments Ethics Committee with the permission number 2021/58).The subjects were randomly divided into 4 groups, and the specified diet and water were given ad libitum . The control group (n = 9) was fed with chow diet for 16 weeks, while the CD (cafeteria diet) group (n = 9) was fed with cafeteria diet for 16 weeks. The subjects in the CD + 7,8-DHF group (n = 11) were given a cafeteria diet for 16 weeks and 7,8-DHF treatment was applied on the last 28 days of the feeding period. 7,8-DHF (Cayman, Michigan USA) was prepared by dissolving in PBS (phosphate buffered saline) containing 17% DMSO (dimethyl sulfoxide) and administered intraperitoneally for 28 days (5 mg/kg/day). In the CD + Vehicle group (n = 11), the subjects were fed with a cafeteria diet for 16 weeks, and PBS containing 17% DMSO, a 7,8-DHF solvent, was given intraperitoneally for the last 28 days of the feeding period (Zeng vd., 2012). The cafeteria diet was created by adding different food items (nut biscuits, fish crackers, fruitcake, potato chips, chocolate wafers) to the chow diet as described previously (Holemans vd. 2004; Zeeni vd. 2015; Buyukdere vd. 2019; Morais Mewes vd. 2019). Based on the energy values specified on the packaging of the foodstuffs used in the calculation of the energy intake, a diet adjustment was made in such a way that 40% came from fats, 55% from carbohydrates and 5% from protein. The ingredients of the cafeteria diet were pulverized in a laboratory grinder. Then, the powder mixture consisting of 70% cafeteria diet and 30% chow diet was turned into a slurry with water and pressed into molds. The feeds, which were prepared by keeping them at 50 ºC for 24 hours, were stored at + 4 ºC. At the end of 16 weeks, the subjects were sacrificed under general anesthesia and kidney tissues were taken. Tissues were stored at -80ºC until the following parameters were studied. MDA determination MDA levels in tissue were studied by thiobarbituric acid reactive substance formation method. Tissue samples were weighed and homogenized in TCA; then, the supernatant was taken, TBA and BHT were added, and the optical density of the samples was read in the spectrophotometer at 535 nm (Buege et al., 1978). GSH determination The modified Ellman method was used for the determination of glutathione in tissue. After the tissue samples were homogenized and centrifuged as in the MDA method, the supernatant was mixed with NaH 2 PO 4 and DTNB solution and incubated for 5–10 minutes at room temperature. The absorbance of the mixture was measured in a spectrophotometer at a wavelength of 412 nm (Aykaç et al., 1985 ). SOD activity The method described by Sun et al. ( 1988 ) was used to determine the SOD activity in tissue and the measurement was performed spectrophotometrically. The method is based on the principle that the superoxide radical, formed by using xanthine and xanthine oxidase, reduces nitroblue tetrazolium (NBT) to form red colored formazone chromogen. SOD activity was measured as a result of spectrophotometric reading of the red color in the medium at a wavelength of 505 nm. CAT activity Catalase is involved in catalyzing the dismutation of hydrogen peroxide to form water and molecular oxygen. In the study, the method developed by Aebi et al. (1984) was used to determine CAT activity. The method is based on the principle of spectrophotometric monitoring of the decrease in H 2 O 2 concentration per unit time at 240 nm (Aebi et al., 1984). NO x determination in tissue : NO x concentration in tissue samples was studied with Griess method (Miranda et al., 2001 ). Tissues were centrifuged at 3500 rpm for 15 minutes after homogenizing with 0.1 M sodium phosphate buffer (pH = 7) (1:9). 0.25 mL of 0.3 M NaOH was added to 500 µL of supernatant. After incubating for 5 minutes at room temperature, an equal amount of VCl3 was added to reduce nitrate in the medium to nitrite and left for 30 minutes incubation at 37°C. Griess I + II reagents were then added, which were mixed in equal amounts. After incubation at 37°C for 10 minutes, the samples were read in spectrophotometer at 540 nm. 6.4 mM stock sodium nitrite (NaNO 2 ) standard was diluted daily and standards were obtained at concentrations of 128, 64, 32, 16, 8, 4, 2 and 1 µM. From the standard curve prepared, the NO x concentration in the samples was calculated as µmol/g tissue. Measurement of renal collagen level The determination of the total amount of collagen (types I-V) in the kidney tissues was performed using a commercial kit according to the manufacturer's instructions (Sircol Collagen Assay Kit, S1111 Rat Std., Biocolor, UK) and as specified by Tsuda et al. ( 2010 ). 0.1 g tissue sample was weighed and kept in pepsin-acetic acid solution overnight and homogenization was performed. 1 mL of Sircol dye was added to 100 µL of homogenate and stirred for 30 minutes, and then centrifuged at 10000 g for 10 minutes. The supernatant was removed and 1 mL of alkaline reagent was added to the remaining pellet and left for 10 minutes. Samples were read in a spectrophotometer at a wavelength of 540 nm. The amount of collagen in the tissues was calculated as µg/mg tissue. Statistical evaluation Results are given as mean ± standard error. Statistical evaluation was performed using Graphpad 4.0 (GraphPad 186 Software, La Jolla, CA, USA). The suitability of the data for normal distribution was tested using the Shapiro-Wilks test. Data analysis was done with one-way analysis of variance, and Tukey test was used for intergroup evaluation and t-test was used for pairwise comparisons. Values with p < 0.05 were considered statistically significant. 3. Results Body weights The initial weights of the mice included in the study were similar among the groups. The beginning body weights of the animals were recorded as 18.69 ± 0.41 grams in the control group, 19.3 ± 0.29 grams in the CD group, 19.61 ± 0.51 grams in the CD + Vehicle group and 18.44 ± 0.57 grams in the CD + 7.8-DHF group. A statistically significant weight gain was observed in body weights of all subjects at the end of 16 weeks. The weights of the subjects increased to 25.67 ± 0.50 grams (p < 0.0001) in the control group, 31.67 ± 0.58 grams (p < 0.0001) in the CD group, 29.1 ± 0.39 grams (p < 0.0001) in the CD + Vehicle group and 27.56 ± 0.68 grams (p < 0.001) in the CD + 7.8-DHF group. While the final body weights of the CD group and CD + Vehicle group fed with cafeteria diet for 16 weeks were significantly higher than the control group (p < 0.0001 and p < 0.01, respectively), there was no significant difference between the CD + 7,8-DHF and control groups (Fig. 1 ). Body weight of CD + Vehicle group subjects (p < 0.05) and CD + 7,8-DHF group subjects (p < 0.001) were found to be statistically significantly lower when compared to CD group (Fig. 1 ). MDA levels The kidney MDA value, which was 39.93 ± 2.15 µmol/g tissue in the control group, increased to 85.89 ± 2.26 µmol/g tissue in the CD group (p < 0.0001). Similar changes were found in the CD + Vehicle group (90.19 ± 1.62 µmol/g tissue, difference from the control p < 0.0001). After 28 days of 7,8-DHF treatment, although the renal MDA level was still higher than the control group (58.78 ± 1.52 µmol/g tissue, difference from the control p < 0.0001), it was found to be lower than the CD and CD + Vehicle groups (CD vs CD + 7, 8-DHF p < 0.0001 and CD + Vehicle vs CD + 7,8-DHF p < 0.0001) (Fig. 2 A). GSH level GSH level in kidney tissue of mice fed with CD for 16 weeks was noticed as 3.69 ± 0.14 µmol/g tissue. This value is statistically significantly lower than the control group (6.06 ± 0.20 µmol/g tissue) (p < 0.0001). In the CD group given 7,8-DHF, the GSH level was found to be 5.30 ± 0.14 µmol/g tissue. Although this value is lower than the control group (difference p < 0.01), it is higher than the CD and CD + Vehicle groups alone (3.91 ± 0.14 µmol/g tissue) (CD vs CD + 7,8-DHF p < 0.0001 and CD + Vehicle vs. CD + 7,8-DHF p < 0.0001) (Fig. 2 B). SOD activity Renal SOD activity, which was 4.17 ± 0.16 units/mg tissue in the control group, decreased to 1.89 ± 0.17 units/mg tissue in the CD group (p < 0.0001). Similar changes were observed in the CD + Vehicle group (1.43 ± 0.13 units/mg tissue, difference from control p < 0.0001). In the CD group receiving 7,8-DHF treatment, renal SOD activity was measured as 3.34 ± 0.12 units/mg tissue. Even though this value is lower than the control group (p < 0.01), it is higher than the CD and CD + Vehicle groups (CD vs CD + 7,8-DHF p < 0.0001 and CD + Vehicle vs CD + 7,8-DHF p < 0.0001) (Fig. 3 A). CAT activity Renal CAT activity of mice in the control group was determined as 24.25 ± 1.15 µmol/H 2 O 2 /min /mg protein. This value decreased to 14.86 ± 0.69 µmol/H 2 O 2 /min/mg protein after 16 weeks of feeding with cafeteria diet (p < 0.0001). A similar decrease was observed in the CD + Vehicle group (15.14 ± 0.67 µmol/H 2 O 2 /min/mg protein, difference from control p < 0.0001). It was noted that renal CAT activity completely restored to control values (24.30 ± 0.81 µmol/H 2 O 2 /min/mg protein) in the CD group receiving 7,8-DHF treatment (CD vs CD + 7,8-DHF p < 0.0001 and CD + Vehicle vs CD + 7.8-DHF p < 0.0001) (Fig. 3 B). NOx level Renal NOx level was found to be 17.62 ± 0.51 nmol/g tissue in the control group, and this value increased to 42.99 ± 1.12 nmol/g tissue in the cafeteria diet group at the end of 16 weeks (p < 0.0001). A similar increase was examined in CD + Vehicle group (42.10 ± 0.91 nmol/g tissue, p < 0.0001). It was seen that NOx values in the CD + 7,8-DHF group that received 7,8-DHF treatment were close to the control data, but were still higher than the control (21.62 ± 0.67 nmol/g tissue, difference from the control p < 0.01). However, the decrease in renal NOx level caused by 7,8-DHF treatment was statistically significant (CD vs CD + 7,8-DHF p < 0.0001 and CD + Vehicle vs CD + 7,8-DHF p < 0.0001) (Fig. 4 ). Collagen level While the renal collagen level was 7.01 ± 0.29 µg/mg tissue in the control group, a significant increase was found in the CD group (12.92 ± 0.45 µg/mg tissue, p < 0.0001). This increament was similar in the CD + Vehicle (12.73 ± 0.51 µg/mg tissue, p < 0.0001) and in the CD + 7.8-DHF groups (13.07 ± 0.62 µg/mg tissue, p < 0.0001). Vehicle or 7,8-DHF administration had no effect in mice on the cafeteria diet. The CD, CD + Vehicle and CD + 7,8-DHF group data, which were close to each other, remained higher than the control group data (Fig. 5 ). 4. Discussion In this study, in which we examined the effects of 7,8-DHF treatment on the kidney in the obesity model created by the cafeteria diet; i) Cafeteria diet induced an increase in oxidative stress, nitrite-nitrate and collagen levels in kidney tissue ii) In cafeteria diet-fed mice, 7,8-DHF treatment for four weeks significantly reduced renal oxidative stress and NOx increase, but did not affect collagen deposition. Both transgenic animal models and diet-induced obesity models have been developed to examine the pathophysiology of obesity at the molecular level (Tschöp and Heiman, 2001). In the cafeteria diet model applied in this study, experimental animals were fed with a variety of human foods (such as nut biscuits, fish crackers, fruit cakes, potato chips, chocolate wafers) that are both high in calorie and delicious (a factor that promotes nutrient intake). The model perfectly mimics the effects of obesity and the metabolic syndrome, as it produces severe obesity, insulin resistance, and significant increase in plasma free fatty acid levels (Holemans vd. 2004; Zeeni vd. 2015; Buyukdere vd. 2019; Morais Mewes vd. 2019). In this study, the development of obesity in mice in the cafeteria diet-fed group is observed from the increase in body weight (Figure 1). Since DMSO was used as the 7,8-DHF carrier in our study, a vehicle group was formed and PBS containing 17% DMSO was given intraperitoneally to this group fed with cafeteria diet. Our results showed that body weight gain in mice in the CD+Vehicle group was greater than that of the control, but less than that of the CD group. This suggests that DMSO may have an effect on reducing weight gain. Indeed, in a study examining the dose-dependent effects of DMSO (0.01%-100%) in 3T3-L1 adipocytes in vitro , clues supporting this view were obtained (Dludla et al., 2018). In the aforementioned study, it has been shown that 10% and higher doses of DMSO reduce the lipid content in adipocytes. It has also been reported that DMSO dose-dependently decreases metabolic activity in adipocytes, causes oxidative stress, and increases the number of necrotic and apoptotic cells. Based on all these data, we think that the less weight gain in the CD+Vehicle group than the CD group may be due to the effects of 17% DMSO on adipocytes. In previous studies, 7,8-DHF has been reported to alleviate weight gain caused by high-fat diet by increasing systemic energy expenditure (Chan et al., 2015; Wood et al., 2018). Supporting former studies, weight gain in the CD+7,8-DHF group was not as much as in the CD group despite the cafeteria diet, and there was no significant difference between the data of this group and the control data. However, the lack of a significant difference between CD+Vehicle and CD+7,8-DHF groups suggested that the effect of 7,8-DHF on body weight was negligible in these study conditions, where cafeteria diet was applied. Complications accompanying obesity include insulin resistance, leptin resistance, decrease in adiponectin, increase in proinflammatory cytokines, RAAS activation, as well as oxidative stress (Tang et al., 2012). Noeman et al (2011) demonstrated the increase in oxidative stress in kidney tissue caused by a 16-week high-fat diet. Researchers have confirmed the existence of oxidative stress with increase in MDA and protein carbonyl, decrease in GSH, decrease in glutathione S transferase, g lutathione peroxidase (GPx) and CAT activities. Pinheiro et al (2018), on the other hand, examined the changes in the renal cortex and medulla separately in rats fed with cafeteria diet for 24 days. The research team showed that GSH depletion and increase in GPx activity are both seen in the cortex and medulla, whereas the increase in lipid peroxidation is more pronounced in the medulla and the decrease of CAT activity is more pronounced in the cortex. In our study, the kidney tissue was examined as a whole without separating it as the cortex-medulla, and the presence of renal oxidative stress was demonstrated by an increase in MDA, a decrease in GSH, and a decrease in SOD and CAT activities (Figure 2 and Figure 3). Oxidative stress is an important factor that plays a role in obesity-related kidney damage, and approaches to reduce oxidative stress prevent this phenomenon to a large extent (Kanthe et al., 2021; Gujjala et al., 2016; Lee et al., 2012). In the obesity model created with the cafeteria diet, it has been observed that the administration of antioxidant diet reduces/prevents oxidative stress-related DNA damage and cell death by reducing oxidative stress in kidney tissue (L E Mballa et al., 2021; La Russa et al., 2019; Leffa et al., 2014). In our study, the effects of 7,8-DHF, a BDNF mimetic and antioxidant molecule, on renal oxidative stress were investigated. Our results show that the increase in MDA, decrease in GSH, decrease in SOD and CAT activities observed in the CD group were significantly alleviated in the group receiving 7,8-DHF treatment, while CAT activity returned completely to control values. DMSO, which is a 7,8-DHF transporter, had no effect on oxidative stress parameters, and the values in this group were not different from the CD group. Thus, the changes recorded in the CD+7,8-DHF group were attributed to the 7,8-DHF itself. These results are consistent with previous in vivo studies reporting that 7,8-DHF treatment has antioxidant effects in hippocampus and liver tissue in a high-fat diet-induced obesity model (Pandey et al., 2020; Kumar et al., 2019). In addition, our findings are in line with previous in vitro studies showing the radical scavenging effect of 7,8-DHF and upregulating antioxidant enzymes such as CAT, Mn-SOD and HO-1 (Choi et al., 2017; Choi et al., 2016). Nitric oxide (NO) is an important paracrine factor that plays a role in the control of both physiological and pathological mechanisms in the cells of the cardiovascular system, nervous system and immune system. It is produced from L-arginine by three different nitric oxide synthase (NOS) enzymes. Of these enzymes, neuronal and endothelial NOS (nNOS and eNOS) are constitutively expressed isoforms. Inducible NOS (iNOS), on the other hand, can be activated in the presence of lipopolysaccharide, cytokines and other agents producing large amounts of NO. Although NO is an important biological mediator, when it is produced excessively by iNOS, it can change the activity and stability of proteins via S-nitrosylation, and also react with superoxide radical to form peroxynitrite, a highly reactive nitrogen species. iNOS activation and excessive NO production are involved in the pathology of inflammatory diseases such as obesity, diabetes, septic shock, atherosclerosis and rheumatoid arthritis (Martin et al., 2018; Aktan, 2004; Förstermann and Sessa, 2012). In our study, nitrite and nitrate levels were measured in kidney tissue as an indicator of NO production. Our results show that the tissue NOx level is increased in the cafeteria diet-fed obese mice compared to the control group. This indicates that obesity increases renal NO production. Findings related to NO production in previous similar obesity studies are contradictory. There are studies revealing that NO production is reduced in obese mice fed a high-fat diet (Gámez-Méndez et al., 2014; Galili et al., 2007). However, there are also articles reporting increased NO production in obesity, similar to our results (Gil-Ortega et al., 2010; Correia-Costa et al., 2016). Although under our operating conditions it is not possible to understand which NOS type is the source of produced NO, we think that the source may be iNOS. Supporting this view, some studies in experimental obesity models reported high iNOS expression and low/unchanged structural NOS (eNOS, nNOS) expression (Noronha et al., 2005; Justo et al., 2013; Tsuchiya et al., 2007; Martin et al., 2018). Studies examining the effect of 7,8-DHF on NO production are limited, with some reporting that 7,8-DHF increases NO production, while others report that it decreases it. For example, Huai et al (2014) showed that 7,8-DHF caused an increase in eNOS-mediated NO production in the vascular endothelium. However, it has also been shown that iNOS, which is activated as a result of high-fat diet and alcohol consumption or lipopolysaccharide stimulation, is suppressed in the presence of 7,8-DHF and iNOS-mediated NO production is reduced (Kumar et al., 2019; Park et al., 2014; Wang et al., 2014). In our study, we observed that the NOx level in the CD+7,8-DHF group was lower than the CD group. Namely, NO production, which was increased by the cafeteria diet, decreased significantly with 7,8-DHF treatment, although it did not return to control values (Figure 4). The absence of such a change in the CD+Vehicle group indicates that the change in the CD+7,8-DHF group is related to 7,8-DHF. Considering the studies reporting iNOS activation in obesity and previous studies showing that 7,8-DHF inhibits iNOS, it may be possible to make the following comment; increased iNOS activity with the cafeteria diet caused excessive NO production in the kidney, and in those mice receiving 7,8-DHF treatment, NO production decreased for iNOS activation was suppressed. However, evaluation of the activity of NOS isoforms, at least iNOS, under these operating conditions is necessary to confirm this interpretation. High-fat diet and obesity cause the kidney to become more susceptible to the development of fibrosis (Declèves and Sharma, 2015). In addition to increased oxidative stress in obesity, increased inflammatory mediators such as TNF-α, IL-6 and adipokines originating from adipose tissue also play a role in the development of kidney damage and fibrosis in obesity (Tang et al., 2012; Wolf and Ziyadeh, 2006). Collagen deposition in tissue is a hallmark of renal fibrosis (Alexakis et al., 2006). Therefore, in our study, renal fibrotic changes were evaluated by examining the accumulation of collagen (type I-V) in the kidney tissue. Our results showed increased collagen deposition in cafeteria diet-fed obese mice, while 7,8-DHF treatment had no additive effect. There is no study in the literature showing the effect of 7,8-DHF on renal fibrosis in any experimental model. However, based on our results, it can be said that 7,8-DHF does not have much effect on the development of renal fibrosis in the cafeteria diet-induced obesity model. In addition, since 7,8-DHF suppresses renal oxidative stress, it is possible to interpret those factors other than oxidative stress (eg, adipose tissue-derived factor) are more important in cafeteria diet-induced renal fibrosis. In this study, it was seen that the cafeteria diet caused an increase in oxidative stress, an increase in NO production and fibrotic changes in the kidney, while 7,8-DHF administration significantly suppressed oxidative stress and NO production. Although the changes in kidney functions with cafeteria diet and 7,8-DHF administration have not been investigated, which is the limiting part of our study, the results of this paper are sufficient to suggest that 7,8-DHF may be protective in obesity-related renal damage. The increasing prevalence of cafeteria diet on eating habits and obesity in the community make these results clinically important. Further studies are needed to examine the renal effects of 7,8-DHF and its effects on other peripheral tissues in more detail. Declarations Contribution of the Authors All authors contributed equally to the work. Conflict of Interest There is no conflict of interest between the authors. References Aebi H. Catalase. Medhods Enzymol. 1984; 105:121-126. Aktan F. iNOS-mediated nitric oxide production and its regulation. Life Sci. 2004;75(6):639-653. Alexakis C, Maxwell P, Bou-Gharios G. Organ-specific collagen expression: implications for renal disease. Nephron Exp Nephrol. 2006;102(3-4):e71-5. Aykaç G, Uysal M, Yalçın AS, et al. The effect of chronic ethanol ingestion on hepatic lipid peroxide, glutathione, glutathione peroxidase and glutathione transferase in rats. Toxicology. 1985; 36: 71-76. Buege A, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978; 52: 302- 310. Buyukdere Y, Gulec A, Akyol A. Cafeteria diet increased adiposity in comparison to high fat diet in young male rats. PeerJ. 2019;7:e6656. Chan CB, Tse MC, Liu X, Zhang S, Schmidt R, Otten R, Liu L, Ye K. Activation of muscular TrkB by its small molecular agonist 7,8-dihydroxyflavone sex-dependently regulates energy metabolism in diet-induced obese mice. Chem Biol. 2015;22(3):355-368. Choi JW, Lee CW, Lee J, Choi DJ, Sohng JK, Park YI. 7,8-Dihydroxyflavone inhibits adipocyte differentiation via antioxidant activity and induces apoptosis in 3T3-L1 preadipocyte cells. Life Sci. 2016;144:103-112. Choi JW, Lee J, Park YI. 7,8-Dihydroxyflavone attenuates TNF-α-induced skin aging in Hs68 human dermal fibroblast cells via down-regulation of the MAPKs/Akt signaling pathways. Biomed Pharmacother. 2017;95:1580-1587. Correia-Costa L, Sousa T, Morato M, Cosme D, Afonso J, Areias JC, Schaefer F, Guerra A, Afonso AC, Azevedo A, Albino-Teixeira A. Oxidative stress and nitric oxide are increased in obese children and correlate with cardiometabolic risk and renal function. Br J Nutr. 2016;116(5):805-815. Declèves AE, Sharma K. Obesity and kidney disease: differential effects of obesity on adipose tissue and kidney inflammation and fibrosis. Curr Opin Nephrol Hypertens. 2015;24(1):28-36. Dludla PV, Jack B, Viraragavan A, Pheiffer C, Johnson R, Louw J, Muller CJF. A dose-dependent effect of dimethyl sulfoxide on lipid content, cell viability and oxidative stress in 3T3-L1 adipocytes. Toxicol Rep. 2018;5:1014-1020. Emili M, Guidi S, Uguagliati B, Giacomini A, Bartesaghi R, Stagni F. Treatment with the flavonoid 7,8-Dihydroxyflavone: a promising strategy for a constellation of body and brain disorders. Crit Rev Food Sci Nutr 2020;1-38. Förstermann U, Sessa WC. Nitric oxide synthases: regulation and function.Eur Heart J. 2012;33(7):829-37, 837a-837d. Galili O, Versari D, Sattler KJ, Olson ML, Mannheim D, McConnell JP, Chade AR, Lerman LO, Lerman A. Early experimental obesity is associated with coronary endothelial dysfunction and oxidative stress. Am J Physiol Heart Circ Physiol. 2007;292(2):H904-911. Gámez-Méndez AM, Vargas-Robles H, Arellano-Mendoza M, Cruz-Laguna E, Rios A, Escalante B. Early stage of obesity potentiates nitric oxide reduction during the development of renal failure. J Nephrol. 2014;27(3):281-287. Gil-Ortega M, Stucchi P, Guzmán-Ruiz R, Cano V, Arribas S, González MC, Ruiz-Gayo M, Fernández-Alfonso MS, Somoza B. Adaptative nitric oxide overproduction in perivascular adipose tissue during early diet-induced obesity. Endocrinology. 2010;151(7):3299-3306. Gray J, Yeo GS, Cox JJ, Morton J, Adlam AL, Keogh JM, Yanovski JA, El Gharbawy A, Han JC, Tung YC, Hodges JR, Raymond FL, O'rahilly S, Farooqi IS. Hyperphagia, severe obesity, impaired cognitive function, and hyperactivity associated with functional loss of one copy of the brain-derived neurotrophic factor (BDNF) gene. Diabetes. 2006;55(12):3366-3371. Gujjala S, Putakala M, Nukala S, Bangeppagari M, Ramaswamy R, Desireddy S. Renoprotective effect of Caralluma fimbriata against high-fat diet-induced oxidative stress in Wistar rats. J Food Drug Anal. 2016;24(3):586-593. Holemans K, Caluwaerts S, Poston L, Van Assche FA. Diet-induced obesity in the rat: a model for gestational diabetes mellitus. Am J Obstet Gynecol. 2004;190(3):858-865. Huai R, Han X, Wang B, Li C, Niu Y, Li R, et al. Vasorelaxing and antihypertensive effects of 7,8-dihydroxyflavone. Am J Hypertens 2014;27(5):750-760. Justo ML, Candiracci M, Dantas AP, de Sotomayor MA, Parrado J, Vila E, Herrera MD, Rodriguez-Rodriguez R. Rice bran enzymatic extract restores endothelial function and vascular contractility in obese rats by reducing vascular inflammation and oxidative stress. J Nutr Biochem. 2013;24(8):1453-1461 Kanthe PS, Patil BS, Das KK. Terminalia arjuna supplementation ameliorates high fat diet-induced oxidative stress in nephrotoxic rats. J Basic Clin Physiol Pharmacol. 2021; 20200106. Kernie SG, Liebl DJ, Parada LF. BDNF regulates eating behavior and locomotor activity in mice. EMBO J. 2000;19(6):1290-1300. Kumar D, Dwivedi DK, Lahkar M, Jangra A. Hepatoprotective potential of 7,8-Dihydroxyflavone against alcohol and high-fat diet induced liver toxicity via attenuation of oxido-nitrosative stress and NF-κB activation. Pharmacol Rep. 2019;71(6):1235-1243. LE Mballa D, Yadang FSA, Tchamgoue AD, Mba JR, Tchokouaha LRY, M Biang E, T Tchinda A, Djomeni Dzeufiet DP, Agbor GA. Cafeteria Diet-Induced Metabolic and Cardiovascular Changes in Rats: The Role of Pişer nigrum Leaf Extract. Evid Based Complement Alternat Med. 2021;2021:5585650. La Russa D, Giordano F, Marrone A, Parafati M, Janda E, Pellegrino D. Oxidative Imbalance and Kidney Damage in Cafeteria Diet-Induced Rat Model of Metabolic Syndrome: Effect of Bergamot Polyphenolic Fraction. Antioxidants (Basel). 2019;8(3):66 Lakkis JI, Weir MR. Obesity and Kidney Disease. Prog Cardiovasc Dis. 2018;61(2):157-167. Lee W, Eom DW, Jung Y, Yamabe N, Lee S, Jeon Y, Hwang YR, Lee JH, Kim YK, Kang KS, Kim SN. Dendrobium moniliforme attenuates high-fat diet-induced renal damage in mice through the regulation of lipid-induced oxidative stress. Am J Chin Med. 2012;40(6):1217-1228. Leffa DD, da Silva J, Daumann F, Dajori AL, Longaretti LM, Damiani AP, de Lira F, Campos F, Ferraz Ade B, Côrrea DS, de Andrade VM. Corrective effects of acerola (Malpighia emarginata DC.) juice intake on biochemical and genotoxical parameters in mice fed on a high-fat diet. Mutat Res. 2014;770:144-152. Manna P, Jain SK. Obesity, oxidative stress, adipose tissue dysfunction, and the associated health risks: causes and therapeutic strategies. Metab Syndr Relat Disord. 2015;13(10):423-444. Martin B, Caron N, Jadot I, Colombaro V, Federici G, Depommier C, Declèves AÉ. Evaluation of inducible nitric oxide synthase inhibition on kidney function and structure in high-fat diet-induced kidney disease. Exp Physiol. 2018;103(1):125-140. Miranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001;5(1):62-71. Morais Mewes J, Rodrigues Silva Gasparin F, Yoshida T, Amâncio Daniel da Silva M, Raquel Marçal Natali M, Francisco Veiga Bizerra P, Sayuri Utsunomiya K, Hideo Gilglioni E, Shigueaki Mito M, Cristiane Mantovanelli G, Thais Lima de Souza B, Makiyama Klosowski E, Luiza Ishii-Iwamoto E, Constantin J, Polimeni Constantin R.: The Role of Mitochondria in Sex-Dependent Differences in Hepatic Steatosis and Oxidative Stress in Response to Cafeteria Diet-Induced Obesity in Mice. Nutrients. 2019;11(7):1618. Noeman SA, Hamooda HE, Baalash AA. Biochemical study of oxidative stress markers in the liver, kidney and heart of high fat diet induced obesity in rats. Diabetol Metab Syndr. 2011;3(1):17. Noronha BT, Li JM, Wheatcroft SB, Shah AM, Kearney MT. Inducible nitric oxide synthase has divergent effects on vascular and metabolic function in obesity. Diabetes. 2005;54(4):1082-1089. Ozata M, Mergen M, Oktenli C, Aydin A, Sanisoglu SY, Bolu E, Yilmaz MI, Sayal A, Isimer A, Ozdemir IC. Increased oxidative stress and hypozincemia in male obesity. Clin Biochem. 2002;35(8):627-631. Pandey SN, Kwatra M, Dwivedi DK, Choubey P, Lahkar M, Jangra A. 7,8-Dihydroxyflavone alleviated the high-fat diet and alcohol-induced memory impairment: behavioral, biochemical and molecular evidence. Psychopharmacology (Berl). 2020;237(6):1827-1840. Park HY, Park C, Hwang HJ, Kim BW, Kim GY, Kim CM, et al. 7,8-Dihydroxyflavone attenuates the release of pro-inflammatory mediators and cytokines in lipopolysaccharide-stimulated BV2 microglial cells through the suppression of the NF-κB and MAPK signaling pathways. Int J Mol Med 2014;33(4):1027-1034. Pellegrino D, La Russa D, Marrone A. Oxidative imbalance and kidney damage: new study perspectives from animal models to hospitalized patients. Antioxidants (Basel). 2019;8(12):594. Pelleymounter MA, Cullen MJ, Wellman CL. Characteristics of BDNF-induced weight loss. Exp Neurol. 1995;131(2):229-238. Pinheiro IRR, Melo MFN, de Sousa SV, Cardoso BG, da Silva TM, Rangel LP, Cortes VF, de Lima Santos H, Chaves VE, Garcia IJP, Barbosa LA. Evaluation of the effect of cafeteria diet on the kidney Na,K-ATPase activity, and oxidative stress. J Cell Biochem. 2019;120(11):19052-19063. Pommer W. Preventive nephrology: the role of obesity in different stages of chronic kidney disease. Kidney Dis (Basel). 2018;4(4):199-204. Sharma K. Obesity and diabetic kidney disease: role of oxidant stress and redox balance. Antioxid Redox Signal. 2016; 25(4): 208–216. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem. 1988; 34: 497-500. Tang J, Yan H, Zhuang S. Inflammation and oxidative stress in obesity-related glomerulopathy. Int J Nephrol. 2012;2012:608397. Tschöp M, Heiman ML. Rodent obesity models: an overview. Exp Clin Endocrinol Diabetes. 2001;109(6):307-319. Tsuchiya K, Sakai H, Suzuki N, Iwashima F, Yoshimoto T, Shichiri M, Hirata Y. Chronic blockade of nitric oxide synthesis reduces adiposity and improves insulin resistance in high fat-induced obese mice. Endocrinology. 2007;148(10):4548-4556. Tsuda K, Nakatani T, Sugama J, Okuwa M, Sanada H. Influence of the timing of switching a protein-free to a protein-containing diet on the wound healing process in a rat all-layer skin defect. Int Wound J. 2010; 7(3), 135-146. Wang B, Wu N, Liang F, Zhang S, Ni W, Cao Y, et al. 7,8-dihydroxyflavone, a small-molecule tropomyosin-related kinase B (TrkB) agonist, attenuates cerebral ischemia and reperfusion injury in rats. J Mol Histol 2014;45(2):129-140. Wang Y, Chen X, Song Y, Caballero B, Cheskin LJ. Association between obesity and kidney disease: a systematic review and meta-analysis. Kidney Int. 2008;73(1):19-33. Wolf G, Ziyadeh FN. Leptin and renal fibrosis. Contrib Nephrol. 2006;151:175-183. Wood J, Tse MCL, Yang X, Brobst D, Liu Z, Pang BPS, Chan WS, Zaw AM, Chow BKC, Ye K, Lee CW, Chan CB. BDNF mimetic alleviates body weight gain in obese mice by enhancing mitochondrial biogenesis in skeletal muscle. Metabolism. 2018;87:113-122. Yeo GS, Connie Hung CC, Rochford J, Keogh J, Gray J, Sivaramakrishnan S, O'Rahilly S, Farooqi IS. A de novo mutation affecting human TrkB associated with severe obesity and developmental delay. Nat Neurosci. 2004;7(11):1187-1189. Zeeni N, Dagher-Hamalian C, Dimassi H, Faour WH. Cafeteria diet-fed mice is a pertinent model of obesity-induced organ damage: a potential role of inflammation. Inflamm Res. 2015;64(7):501-512. Zeng Y, Lv F, Li L, Yu H, Dong M, Fu Q. 7,8-dihydroxyflavone rescues spatial memory and synaptic plasticity in cognitively impaired aged rats. J Neurochem. 2012;122(4):800-811. Zhao P, Li X, Li Y, Zhu J, Sun Y, Hong J. Mechanism of miR-365 in regulating BDNF-TrkB signal axis of HFD/STZ induced diabetic nephropathy fibrosis and renal function. Int Urol Nephrol. 2021;53(10):2177-2187. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2053626","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":135935162,"identity":"dd718f09-4fbc-47d6-ac6f-57e10d9dee2a","order_by":0,"name":"Emine Gulceri Gulec Peker","email":"data:image/png;base64,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","orcid":"","institution":"Giresun University","correspondingAuthor":true,"prefix":"","firstName":"Emine","middleName":"Gulceri Gulec","lastName":"Peker","suffix":""},{"id":135935163,"identity":"acbdea67-ba42-4212-89ee-7da1c63a2256","order_by":1,"name":"Selma Cirrik","email":"","orcid":"","institution":"Ordu University","correspondingAuthor":false,"prefix":"","firstName":"Selma","middleName":"","lastName":"Cirrik","suffix":""},{"id":135935164,"identity":"885ccc1c-fdf9-406f-ac5e-160298e3da96","order_by":2,"name":"Gulay Hacioglu","email":"","orcid":"","institution":"Giresun University","correspondingAuthor":false,"prefix":"","firstName":"Gulay","middleName":"","lastName":"Hacioglu","suffix":""},{"id":135935165,"identity":"33665fd1-a64d-455f-9828-249a61ba6d01","order_by":3,"name":"Elif Sahin","email":"","orcid":"","institution":"Karadeniz Technical University","correspondingAuthor":false,"prefix":"","firstName":"Elif","middleName":"","lastName":"Sahin","suffix":""},{"id":135935166,"identity":"b9271e95-6059-45db-97f2-64d8b592414d","order_by":4,"name":"Ahmet Alver","email":"","orcid":"","institution":"Karadeniz Technical University","correspondingAuthor":false,"prefix":"","firstName":"Ahmet","middleName":"","lastName":"Alver","suffix":""}],"badges":[],"createdAt":"2022-09-11 11:59:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2053626/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2053626/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":26469466,"identity":"16fe25ca-bbb9-4b88-8ac4-167d5cc4be76","added_by":"auto","created_at":"2022-09-14 20:49:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":30800,"visible":true,"origin":"","legend":"\u003cp\u003eBody weight values in all groups at the end of 16 weeks. Difference from control group *p\u0026lt;0.01, **p\u0026lt;0.0001; difference from CD group #p\u0026lt;0.05, #p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-2053626/v1/37347940af485cd32473fc43.png"},{"id":26469467,"identity":"9c9f1e61-245e-4d60-9168-7ef960a4093a","added_by":"auto","created_at":"2022-09-14 20:49:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":33385,"visible":true,"origin":"","legend":"\u003cp\u003eRenal MDA (A) and GSH (B) levels in all groups. Difference from control group *p\u0026lt;0.001, **p\u0026lt;0.0001; difference from CD group #p\u0026lt;0.0001; difference from CD+Vehicle group fp \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-2053626/v1/90855033ec24e028413b3dd1.png"},{"id":26471101,"identity":"eadd892d-230e-4e9c-aa51-815d6f308526","added_by":"auto","created_at":"2022-09-14 20:54:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":67834,"visible":true,"origin":"","legend":"\u003cp\u003eRenal SOD (A) and CAT (B) activity in all groups. Difference from control group *p\u0026lt;0.01, **p\u0026lt;0.0001; difference from CD group #p\u0026lt;0.0001; difference from CD+Vehicle group fp \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-2053626/v1/b5d921f3203412a9c5914205.png"},{"id":26469469,"identity":"f47cce18-ca3b-4515-a6ec-f62f5c025a0c","added_by":"auto","created_at":"2022-09-14 20:49:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":37920,"visible":true,"origin":"","legend":"\u003cp\u003eRenal nitrite-nitrate (NOx) level in all groups. Difference from control group *p\u0026lt;0.01, **p\u0026lt;0.0001; difference from CD group #p\u0026lt;0.0001; difference from CD+Vehicle group fp \u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-2053626/v1/5acf89ed7bb4cd0bab633ef4.png"},{"id":26471100,"identity":"ee333582-dbc1-4aa2-910f-6f27adbfbc97","added_by":"auto","created_at":"2022-09-14 20:54:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":34103,"visible":true,"origin":"","legend":"\u003cp\u003eRenal collagen level in the groups. Difference from control group *p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-2053626/v1/35e5c1a361905c1c08a1871d.png"},{"id":26522077,"identity":"e72a67a9-cb72-41aa-afb0-bf1a7f8f6698","added_by":"auto","created_at":"2022-09-15 18:59:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":451842,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2053626/v1/8d6707cc-f311-4a84-a3bb-36f5cf7028ee.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Renal effects of 7,8-Dihydroxyflavone in cafeteria diet-induced obesity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eObesity is an important risk factor for chronic kidney disease as well as cardiovascular diseases and diabetes mellitus. Some changes that occur with obesity, for example, increase in plasma free fatty acids, hyperinsulinemia, insulin resistance, oxidative stress, proinflammatory conditions, decrease in adiponectin, leptin resistance, and activation of the renin-angiotensin-aldosterone system (RAAS) cause glomerular and tubular damage, inducing chronic predisposes for kidney disease (Pommer, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Manna and Jain, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Wang et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Hyperinsulinemia contributes to this process by generating endothelial dysfunction, increased oxidative stress, and pro-inflammatory and pro-fibrotic changes in the kidney. In addition, renal lipotoxicity, which occurs with the accumulation of triglycerides in the glomeruli and proximal tubules, is an important factor in kidney damage. Obesity-related glomerulopathy (ORG) is an important clinical manifestation of all these changes (Lakkis and Weir, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIncreased oxidative stress is accepted as one of the important mechanisms in the pathophysiology of obesity. The main factors that stimulate an increase in oxidative stress in obesity are hyperglycemia and the change in cell metabolism due to the increase in plasma free fatty acids and the increase in the production of reactive oxygen species (ROS) (Manna and Jain, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Sharma, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pellegrino et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, some proinflammatory cytokines such as TNF-α and IL-6 released from adipose tissue contribute to the increment in oxidative stress by activating NADPH oxidase (NOX). Another factor originating from adipose tissue that activates NOX is the hormone leptin, which is why there is hyperleptinemia in obese individuals (Manna and Jain, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Sharma, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Pellegrino et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These adipose tissue-derived factors (TNF-α, IL-6 and leptin) also cause the kidney to become more susceptible for the development of fibrosis (Decl\u0026egrave;ves and Sharma, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Tang et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Wolf and Ziyadeh, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In addition to the increase in ROS production, the weakening of antioxidant activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) also contributes to the enlargement in oxidative damage in obesity (Ozata et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Noeman et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). It has been shown that approaches to reduce oxidative stress largely prevent renal damage observed in obesity. For example, renal oxidative stress, glomerular-tubular damage and functional loss that occur in experimental obesity models induced by high-fat diet are significantly abolished in the presence of herbal extracts with known antioxidant effects such as \u003cem\u003eTerminalia arjuna\u003c/em\u003e, \u003cem\u003eCaralluma fimbriata\u003c/em\u003e, and \u003cem\u003eDendrobium moniliforme\u003c/em\u003e (Kanthe et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Gujjala et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e7,8-Dihydroxyflavone (7,8-DHF) is a natural flavonoid found in the leaves of \u003cem\u003eGodmania aesculifolia\u003c/em\u003e, \u003cem\u003eTridax procumbens\u003c/em\u003e and \u003cem\u003eprimula\u003c/em\u003e tree. \u003cem\u003eIn vitro\u003c/em\u003e studies have shown that the molecule has radical scavenging effects and up-regulates antioxidant enzymes such as CAT, SOD and hemoxygenase-1 (HO-1) (Choi et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Choi et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). It has even been shown \u003cem\u003ein vitro\u003c/em\u003e that the production of proinflammatory mediators (eg, nitric oxide, prostaglandin E2, interleukin 1β) caused by lipopolysaccharide induction is inhibited in the presence of 7,8-DHF (Park et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The most important feature of 7,8-DHF is that it can mimic the effects of brain-derived neurotrophic factor (BDNF), which is one of the major factors playing a role in neuron development, vitality and maintenance of normal cell functions. The molecule shows this effect by binding to the tyrosine receptor kinase B (TrkB), the main receptor of BDNF. Indeed, the attenuating effects of 7,8-DHF have been demonstrated in some diseases involving BDNF, such as Alzheimer's, Parkinson's, depression, and obesity (Emili et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe BDNF/TrkB pathway plays an important role in the regulation of body weight. In rats, BDNF infusion into the lateral ventricle suppresses food intake and body weight gain (Pelleymounter et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). Ablation or mutation of this pathway results in hyperphagia and obesity (Gray et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e, Yeo et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2004\u003c/span\u003e, Kernie et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). In addition, it has been presented that 7,8-DHF administration increases systemic energy consumption by stimulating uncoupling protein 1 (UCP1) expression and AMP-activated protein kinase (AMPK) activity in skeletal muscle, thus preventing the development of obesity. The lack of anti-obesity effect of 7,8-DHF in muscular TrkB-receptor knockout mice suggests that this effect is mediated by muscular TrkB receptors, not hypothalamus (Chan et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wood et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As a result, it is clear that not only the central activation of the BDNF/TrkB pathway, but also its peripheral activation participates in the regulation of body weight.\u003c/p\u003e \u003cp\u003eAmong the obesity studies, \u003cem\u003ein vivo\u003c/em\u003e studies displaying the effects of 7,8-DHF on the increase in oxidative stress are limited. In these studies, it was indicated that the increase in oxidative stress caused by high-fat diet in the hippocampus and liver was abolished by 7,8-DHF treatment in 4 weeks (Pandey et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kumar et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Kidney tissue, on the other hand, has not been studied before in obesity, in terms of the effects of BDNF/TrkB pathway or 7,8-DHF. Based on previous studies showing that approaches to prevent the increase in oxidative stress reduce obesity-related renal damage, 7,8-DHF administration is also likely to have positive effects on the kidneys. Thus, in our study, we aimed to examine the renal effects of 7,8-DHF treatment in obese mice. In the experimental obesity model selected in this study, the mice were fed with both high-calorie and delicious human food, which is defined as the cafeteria diet, such as hazelnut biscuits, fish crackers, fruitcakes, potato chips and chocolate wafers. Since this type of nutrition and obesity is an increasingly common problem and obesity is an important risk factor for chronic kidney disease, we think it is important to reveal the effects of 7,8-DHF, which is known for its anti-obesity effects and antioxidant effects on the kidneys.\u003c/p\u003e"},{"header":"2. Material And Method","content":"\u003cp\u003e \u003cstrong\u003eExperimental animals and grouping\u003c/strong\u003e \u003cp\u003eIn the study, 4\u0026ndash;5 weeks old (17\u0026ndash;20 g) male C57BL/6 mice were used. Experimental procedures were approved by the Local Ethics Committee for animal experiments \u003cb\u003e(\u003c/b\u003eKaradeniz Technical University Animal Experiments Ethics Committee with the permission number 2021/58).The subjects were randomly divided into 4 groups, and the specified diet and water were given \u003cem\u003ead libitum\u003c/em\u003e. The control group (n\u0026thinsp;=\u0026thinsp;9) was fed with chow diet for 16 weeks, while the CD (cafeteria diet) group (n\u0026thinsp;=\u0026thinsp;9) was fed with cafeteria diet for 16 weeks. The subjects in the CD\u0026thinsp;+\u0026thinsp;7,8-DHF group (n\u0026thinsp;=\u0026thinsp;11) were given a cafeteria diet for 16 weeks and 7,8-DHF treatment was applied on the last 28 days of the feeding period. 7,8-DHF (Cayman, Michigan USA) was prepared by dissolving in PBS (phosphate buffered saline) containing 17% DMSO (dimethyl sulfoxide) and administered intraperitoneally for 28 days (5 mg/kg/day). In the CD\u0026thinsp;+\u0026thinsp;Vehicle group (n\u0026thinsp;=\u0026thinsp;11), the subjects were fed with a cafeteria diet for 16 weeks, and PBS containing 17% DMSO, a 7,8-DHF solvent, was given intraperitoneally for the last 28 days of the feeding period (Zeng vd., 2012).\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThe cafeteria diet was created by adding different food items (nut biscuits, fish crackers, fruitcake, potato chips, chocolate wafers) to the chow diet as described previously (Holemans vd. 2004; Zeeni vd. 2015; Buyukdere vd. 2019; Morais Mewes vd. 2019). Based on the energy values specified on the packaging of the foodstuffs used in the calculation of the energy intake, a diet adjustment was made in such a way that 40% came from fats, 55% from carbohydrates and 5% from protein. The ingredients of the cafeteria diet were pulverized in a laboratory grinder. Then, the powder mixture consisting of 70% cafeteria diet and 30% chow diet was turned into a slurry with water and pressed into molds. The feeds, which were prepared by keeping them at 50 \u0026ordm;C for 24 hours, were stored at +\u0026thinsp;4 \u0026ordm;C.\u003c/p\u003e \u003cp\u003eAt the end of 16 weeks, the subjects were sacrificed under general anesthesia and kidney tissues were taken. Tissues were stored at -80\u0026ordm;C until the following parameters were studied.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMDA determination\u003c/strong\u003e \u003cp\u003eMDA levels in tissue were studied by thiobarbituric acid reactive substance formation method. Tissue samples were weighed and homogenized in TCA; then, the supernatant was taken, TBA and BHT were added, and the optical density of the samples was read in the spectrophotometer at 535 nm (Buege et al., 1978).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGSH determination\u003c/strong\u003e \u003cp\u003eThe modified Ellman method was used for the determination of glutathione in tissue. After the tissue samples were homogenized and centrifuged as in the MDA method, the supernatant was mixed with NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e and DTNB solution and incubated for 5\u0026ndash;10 minutes at room temperature. The absorbance of the mixture was measured in a spectrophotometer at a wavelength of 412 nm (Ayka\u0026ccedil; et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1985\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSOD activity\u003c/strong\u003e \u003cp\u003eThe method described by Sun et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) was used to determine the SOD activity in tissue and the measurement was performed spectrophotometrically. The method is based on the principle that the superoxide radical, formed by using xanthine and xanthine oxidase, reduces nitroblue tetrazolium (NBT) to form red colored formazone chromogen. SOD activity was measured as a result of spectrophotometric reading of the red color in the medium at a wavelength of 505 nm.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCAT activity\u003c/strong\u003e \u003cp\u003eCatalase is involved in catalyzing the dismutation of hydrogen peroxide to form water and molecular oxygen. In the study, the method developed by Aebi et al. (1984) was used to determine CAT activity. The method is based on the principle of spectrophotometric monitoring of the decrease in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration per unit time at 240 nm (Aebi et al., 1984).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eNO\u003c/b\u003e \u003csub\u003e \u003cb\u003ex\u003c/b\u003e \u003c/sub\u003e \u003cb\u003edetermination in tissue\u003c/b\u003e: NO\u003csub\u003ex\u003c/sub\u003e concentration in tissue samples was studied with Griess method (Miranda et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Tissues were centrifuged at 3500 rpm for 15 minutes after homogenizing with 0.1 M sodium phosphate buffer (pH\u0026thinsp;=\u0026thinsp;7) (1:9). 0.25 mL of 0.3 M NaOH was added to 500 \u0026micro;L of supernatant. After incubating for 5 minutes at room temperature, an equal amount of VCl3 was added to reduce nitrate in the medium to nitrite and left for 30 minutes incubation at 37\u0026deg;C. Griess I\u0026thinsp;+\u0026thinsp;II reagents were then added, which were mixed in equal amounts. After incubation at 37\u0026deg;C for 10 minutes, the samples were read in spectrophotometer at 540 nm. 6.4 mM stock sodium nitrite (NaNO\u003csub\u003e2\u003c/sub\u003e) standard was diluted daily and standards were obtained at concentrations of 128, 64, 32, 16, 8, 4, 2 and 1 \u0026micro;M. From the standard curve prepared, the NO\u003csub\u003ex\u003c/sub\u003e concentration in the samples was calculated as \u0026micro;mol/g tissue.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMeasurement of renal collagen level\u003c/strong\u003e \u003cp\u003eThe determination of the total amount of collagen (types I-V) in the kidney tissues was performed using a commercial kit according to the manufacturer's instructions (Sircol Collagen Assay Kit, S1111 Rat Std., Biocolor, UK) and as specified by Tsuda et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). 0.1 g tissue sample was weighed and kept in pepsin-acetic acid solution overnight and homogenization was performed. 1 mL of Sircol dye was added to 100 \u0026micro;L of homogenate and stirred for 30 minutes, and then centrifuged at 10000 g for 10 minutes. The supernatant was removed and 1 mL of alkaline reagent was added to the remaining pellet and left for 10 minutes. Samples were read in a spectrophotometer at a wavelength of 540 nm. The amount of collagen in the tissues was calculated as \u0026micro;g/mg tissue.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical evaluation\u003c/strong\u003e \u003cp\u003eResults are given as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error. Statistical evaluation was performed using Graphpad 4.0 (GraphPad 186 Software, La Jolla, CA, USA). The suitability of the data for normal distribution was tested using the Shapiro-Wilks test. Data analysis was done with one-way analysis of variance, and Tukey test was used for intergroup evaluation and t-test was used for pairwise comparisons. Values with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003e \u003cstrong\u003eBody weights\u003c/strong\u003e \u003cp\u003eThe initial weights of the mice included in the study were similar among the groups. The beginning body weights of the animals were recorded as 18.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41 grams in the control group, 19.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 grams in the CD group, 19.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 grams in the CD\u0026thinsp;+\u0026thinsp;Vehicle group and 18.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 grams in the CD\u0026thinsp;+\u0026thinsp;7.8-DHF group. A statistically significant weight gain was observed in body weights of all subjects at the end of 16 weeks. The weights of the subjects increased to 25.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.50 grams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the control group, 31.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 grams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the CD group, 29.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39 grams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the CD\u0026thinsp;+\u0026thinsp;Vehicle group and 27.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.68 grams (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the CD\u0026thinsp;+\u0026thinsp;7.8-DHF group. While the final body weights of the CD group and CD\u0026thinsp;+\u0026thinsp;Vehicle group fed with cafeteria diet for 16 weeks were significantly higher than the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively), there was no significant difference between the CD\u0026thinsp;+\u0026thinsp;7,8-DHF and control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Body weight of CD\u0026thinsp;+\u0026thinsp;Vehicle group subjects (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and CD\u0026thinsp;+\u0026thinsp;7,8-DHF group subjects (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were found to be statistically significantly lower when compared to CD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eMDA levels\u003c/strong\u003e \u003cp\u003eThe kidney MDA value, which was 39.93\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15 \u0026micro;mol/g tissue in the control group, increased to 85.89\u0026thinsp;\u0026plusmn;\u0026thinsp;2.26 \u0026micro;mol/g tissue in the CD group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Similar changes were found in the CD\u0026thinsp;+\u0026thinsp;Vehicle group (90.19\u0026thinsp;\u0026plusmn;\u0026thinsp;1.62 \u0026micro;mol/g tissue, difference from the control p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). After 28 days of 7,8-DHF treatment, although the renal MDA level was still higher than the control group (58.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52 \u0026micro;mol/g tissue, difference from the control p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), it was found to be lower than the CD and CD\u0026thinsp;+\u0026thinsp;Vehicle groups (CD vs CD\u0026thinsp;+\u0026thinsp;7, 8-DHF p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and CD\u0026thinsp;+\u0026thinsp;Vehicle vs CD\u0026thinsp;+\u0026thinsp;7,8-DHF p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGSH level\u003c/strong\u003e \u003cp\u003eGSH level in kidney tissue of mice fed with CD for 16 weeks was noticed as 3.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u0026micro;mol/g tissue. This value is statistically significantly lower than the control group (6.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 \u0026micro;mol/g tissue) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In the CD group given 7,8-DHF, the GSH level was found to be 5.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u0026micro;mol/g tissue. Although this value is lower than the control group (difference p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), it is higher than the CD and CD\u0026thinsp;+\u0026thinsp;Vehicle groups alone (3.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 \u0026micro;mol/g tissue) (CD vs CD\u0026thinsp;+\u0026thinsp;7,8-DHF p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and CD\u0026thinsp;+\u0026thinsp;Vehicle vs. CD\u0026thinsp;+\u0026thinsp;7,8-DHF p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSOD activity\u003c/strong\u003e \u003cp\u003eRenal SOD activity, which was 4.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 units/mg tissue in the control group, decreased to 1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 units/mg tissue in the CD group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Similar changes were observed in the CD\u0026thinsp;+\u0026thinsp;Vehicle group (1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 units/mg tissue, difference from control p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). In the CD group receiving 7,8-DHF treatment, renal SOD activity was measured as 3.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12 units/mg tissue. Even though this value is lower than the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), it is higher than the CD and CD\u0026thinsp;+\u0026thinsp;Vehicle groups (CD vs CD\u0026thinsp;+\u0026thinsp;7,8-DHF p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and CD\u0026thinsp;+\u0026thinsp;Vehicle vs CD\u0026thinsp;+\u0026thinsp;7,8-DHF p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCAT activity\u003c/strong\u003e \u003cp\u003eRenal CAT activity of mice in the control group was determined as 24.25\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15 \u0026micro;mol/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min /mg protein. This value decreased to 14.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69 \u0026micro;mol/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min/mg protein after 16 weeks of feeding with cafeteria diet (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). A similar decrease was observed in the CD\u0026thinsp;+\u0026thinsp;Vehicle group (15.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 \u0026micro;mol/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min/mg protein, difference from control p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). It was noted that renal CAT activity completely restored to control values (24.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 \u0026micro;mol/H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e/min/mg protein) in the CD group receiving 7,8-DHF treatment (CD vs CD\u0026thinsp;+\u0026thinsp;7,8-DHF p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and CD\u0026thinsp;+\u0026thinsp;Vehicle vs CD\u0026thinsp;+\u0026thinsp;7.8-DHF p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNOx level\u003c/strong\u003e \u003cp\u003eRenal NOx level was found to be 17.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 nmol/g tissue in the control group, and this value increased to 42.99\u0026thinsp;\u0026plusmn;\u0026thinsp;1.12 nmol/g tissue in the cafeteria diet group at the end of 16 weeks (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). A similar increase was examined in CD\u0026thinsp;+\u0026thinsp;Vehicle group (42.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91 nmol/g tissue, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). It was seen that NOx values in the CD\u0026thinsp;+\u0026thinsp;7,8-DHF group that received 7,8-DHF treatment were close to the control data, but were still higher than the control (21.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 nmol/g tissue, difference from the control p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, the decrease in renal NOx level caused by 7,8-DHF treatment was statistically significant (CD vs CD\u0026thinsp;+\u0026thinsp;7,8-DHF p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and CD\u0026thinsp;+\u0026thinsp;Vehicle vs CD\u0026thinsp;+\u0026thinsp;7,8-DHF p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCollagen level\u003c/strong\u003e \u003cp\u003eWhile the renal collagen level was 7.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 \u0026micro;g/mg tissue in the control group, a significant increase was found in the CD group (12.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45 \u0026micro;g/mg tissue, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). This increament was similar in the CD\u0026thinsp;+\u0026thinsp;Vehicle (12.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 \u0026micro;g/mg tissue, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and in the CD\u0026thinsp;+\u0026thinsp;7.8-DHF groups (13.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 \u0026micro;g/mg tissue, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Vehicle or 7,8-DHF administration had no effect in mice on the cafeteria diet. The CD, CD\u0026thinsp;+\u0026thinsp;Vehicle and CD\u0026thinsp;+\u0026thinsp;7,8-DHF group data, which were close to each other, remained higher than the control group data (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, in which we examined the effects of 7,8-DHF treatment on the kidney in the obesity model created by the cafeteria diet; \u003cstrong\u003ei)\u003c/strong\u003e Cafeteria diet induced an increase in oxidative stress, nitrite-nitrate and collagen levels in kidney tissue \u003cstrong\u003eii)\u003c/strong\u003e In cafeteria diet-fed mice, 7,8-DHF treatment for four weeks significantly reduced renal oxidative stress and NOx increase, but did not affect collagen deposition.\u003c/p\u003e\n\u003cp\u003eBoth transgenic animal models and diet-induced obesity models have been developed to examine the pathophysiology of obesity at the molecular level (Tsch\u0026ouml;p and Heiman, 2001). In the cafeteria diet model applied in this study, experimental animals were fed with a variety of human foods (such as nut biscuits, fish crackers, fruit cakes, potato chips, chocolate wafers) that are both high in calorie and delicious (a factor that promotes nutrient intake). The model perfectly mimics the effects of obesity and the metabolic syndrome, as it produces severe obesity, insulin resistance, and significant increase in plasma free fatty acid levels (Holemans vd. 2004; Zeeni vd. 2015; Buyukdere vd. 2019; Morais Mewes vd. 2019). In this study, the development of obesity in mice in the cafeteria diet-fed group is observed from the increase in body weight (Figure 1). Since DMSO was used as the 7,8-DHF carrier in our study, a vehicle group was formed and PBS containing 17% DMSO was given intraperitoneally to this group fed with cafeteria diet. Our results showed that body weight gain in mice in the CD+Vehicle group was greater than that of the control, but less than that of the CD group. This suggests that DMSO may have an effect on reducing weight gain. Indeed, in a study examining the dose-dependent effects of DMSO (0.01%-100%) in 3T3-L1 adipocytes \u003cem\u003ein vitro\u003c/em\u003e, clues supporting this view were obtained (Dludla et al., 2018). In the aforementioned study, it has been shown that 10% and higher doses of DMSO reduce the lipid content in adipocytes. It has also been reported that DMSO dose-dependently decreases metabolic activity in adipocytes, causes oxidative stress, and increases the number of necrotic and apoptotic cells. Based on all these data, we think that the less weight gain in the CD+Vehicle group than the CD group may be due to the effects of 17% DMSO on adipocytes.\u003c/p\u003e\n\u003cp\u003eIn previous studies, 7,8-DHF has been reported to alleviate weight gain caused by high-fat diet by increasing systemic energy expenditure (Chan et al., 2015; Wood et al., 2018). Supporting former studies, weight gain in the CD+7,8-DHF group was not as much as in the CD group despite the cafeteria diet, and there was no significant difference between the data of this group and the control data. However, the lack of a significant difference between CD+Vehicle and CD+7,8-DHF groups suggested that the effect of 7,8-DHF on body weight was negligible in these study conditions, where cafeteria diet was applied.\u003c/p\u003e\n\u003cp\u003eComplications accompanying obesity include insulin resistance, leptin resistance, decrease in adiponectin, increase in proinflammatory cytokines, RAAS activation, as well as oxidative stress (Tang et al., 2012). Noeman et al (2011) demonstrated the increase in oxidative stress in kidney tissue caused by a 16-week high-fat diet. Researchers have confirmed the existence of oxidative stress with increase in MDA and protein carbonyl, decrease in GSH, decrease in glutathione S transferase, g lutathione peroxidase (GPx) and CAT activities. Pinheiro et al (2018), on the other hand, examined the changes in the renal cortex and medulla separately in rats fed with cafeteria diet for 24 days. The research team showed that GSH depletion and increase in GPx activity are both seen in the cortex and medulla, whereas the increase in lipid peroxidation is more pronounced in the medulla and the decrease of CAT activity is more pronounced in the cortex. In our study, the kidney tissue was examined as a whole without separating it as the cortex-medulla, and the presence of renal oxidative stress was demonstrated by an increase in MDA, a decrease in GSH, and a decrease in SOD and CAT activities (Figure 2 and Figure 3).\u003c/p\u003e\n\u003cp\u003eOxidative stress is an important factor that plays a role in obesity-related kidney damage, and approaches to reduce oxidative stress prevent this phenomenon to a large extent (Kanthe et al., 2021; Gujjala et al., 2016; Lee et al., 2012). In the obesity model created with the cafeteria diet, it has been observed that the administration of antioxidant diet reduces/prevents oxidative stress-related DNA damage and cell death by reducing oxidative stress in kidney tissue (L E Mballa et al., 2021; La Russa et al., 2019; Leffa et al., 2014). In our study, the effects of 7,8-DHF, a BDNF mimetic and antioxidant molecule, on renal oxidative stress were investigated. Our results show that the increase in MDA, decrease in GSH, decrease in SOD and CAT activities observed in the CD group were significantly alleviated in the group receiving 7,8-DHF treatment, while CAT activity returned completely to control values. DMSO, which is a 7,8-DHF transporter, had no effect on oxidative stress parameters, and the values in this group were not different from the CD group. Thus, the changes recorded in the CD+7,8-DHF group were attributed to the 7,8-DHF itself. These results are consistent with previous \u003cem\u003ein vivo\u003c/em\u003e studies reporting that 7,8-DHF treatment has antioxidant effects in hippocampus and liver tissue in a high-fat diet-induced obesity model (Pandey et al., 2020; Kumar et al., 2019). In addition, our findings are in line with previous \u003cem\u003ein vitro\u003c/em\u003e studies showing the radical scavenging effect of 7,8-DHF and upregulating antioxidant enzymes such as CAT, Mn-SOD and HO-1 (Choi et al., 2017; Choi et al., 2016).\u003c/p\u003e\n\u003cp\u003eNitric oxide (NO) is an important paracrine factor that plays a role in the control of both physiological and pathological mechanisms in the cells of the cardiovascular system, nervous system and immune system. It is produced from L-arginine by three different nitric oxide synthase (NOS) enzymes. Of these enzymes, neuronal and endothelial NOS (nNOS and eNOS) are constitutively expressed isoforms. Inducible NOS (iNOS), on the other hand, can be activated in the presence of lipopolysaccharide, cytokines and other agents producing large amounts of NO. Although NO is an important biological mediator, when it is produced excessively by iNOS, it can change the activity and stability of proteins via S-nitrosylation, and also react with superoxide radical to form peroxynitrite, a highly reactive nitrogen species. iNOS activation and excessive NO production are involved in the pathology of inflammatory diseases such as obesity, diabetes, septic shock, atherosclerosis and rheumatoid arthritis (Martin et al., 2018; Aktan, 2004; F\u0026ouml;rstermann and Sessa, 2012).\u003c/p\u003e\n\u003cp\u003eIn our study, nitrite and nitrate levels were measured in kidney tissue as an indicator of NO production. Our results show that the tissue NOx level is increased in the cafeteria diet-fed obese mice compared to the control group. This indicates that obesity increases renal NO production. Findings related to NO production in previous similar obesity studies are contradictory. There are studies revealing that NO production is reduced in obese mice fed a high-fat diet (G\u0026aacute;mez-M\u0026eacute;ndez et al., 2014; Galili et al., 2007). However, there are also articles reporting increased NO production in obesity, similar to our results (Gil-Ortega et al., 2010; Correia-Costa et al., 2016). Although under our operating conditions it is not possible to understand which NOS type is the source of produced NO, we think that the source may be iNOS. Supporting this view, some studies in experimental obesity models reported high iNOS expression and low/unchanged structural NOS (eNOS, nNOS) expression (Noronha et al., 2005; Justo et al., 2013; Tsuchiya et al., 2007; Martin et al., 2018). Studies examining the effect of 7,8-DHF on NO production are limited, with some reporting that 7,8-DHF increases NO production, while others report that it decreases it. For example, Huai et al (2014) showed that 7,8-DHF caused an increase in eNOS-mediated NO production in the vascular endothelium. However, it has also been shown that iNOS, which is activated as a result of high-fat diet and alcohol consumption or lipopolysaccharide stimulation, is suppressed in the presence of 7,8-DHF and iNOS-mediated NO production is reduced (Kumar et al., 2019; Park et al., 2014; Wang et al., 2014). In our study, we observed that the NOx level in the CD+7,8-DHF group was lower than the CD group. Namely, NO production, which was increased by the cafeteria diet, decreased significantly with 7,8-DHF treatment, although it did not return to control values (Figure 4). The absence of such a change in the CD+Vehicle group indicates that the change in the CD+7,8-DHF group is related to 7,8-DHF. Considering the studies reporting iNOS activation in obesity and previous studies showing that 7,8-DHF inhibits iNOS, it may be possible to make the following comment; increased iNOS activity with the cafeteria diet caused excessive NO production in the kidney, and in those mice receiving 7,8-DHF treatment, NO production decreased for iNOS activation was suppressed. However, evaluation of the activity of NOS isoforms, at least iNOS, under these operating conditions is necessary to confirm this interpretation.\u003c/p\u003e\n\u003cp\u003eHigh-fat diet and obesity cause the kidney to become more susceptible to the development of fibrosis (Decl\u0026egrave;ves and Sharma, 2015). In addition to increased oxidative stress in obesity, increased inflammatory mediators such as TNF-\u0026alpha;, IL-6 and adipokines originating from adipose tissue also play a role in the development of kidney damage and fibrosis in obesity (Tang et al., 2012; Wolf and Ziyadeh, 2006). Collagen deposition in tissue is a hallmark of renal fibrosis (Alexakis et al., 2006). Therefore, in our study, renal fibrotic changes were evaluated by examining the accumulation of collagen (type I-V) in the kidney tissue. Our results showed increased collagen deposition in cafeteria diet-fed obese mice, while 7,8-DHF treatment had no additive effect. There is no study in the literature showing the effect of 7,8-DHF on renal fibrosis in any experimental model. However, based on our results, it can be said that 7,8-DHF does not have much effect on the development of renal fibrosis in the cafeteria diet-induced obesity model. In addition, since 7,8-DHF suppresses renal oxidative stress, it is possible to interpret those factors other than oxidative stress (eg, adipose tissue-derived factor) are more important in cafeteria diet-induced renal fibrosis.\u003c/p\u003e\n\u003cp\u003eIn this study, it was seen that the cafeteria diet caused an increase in oxidative stress, an increase in NO production and fibrotic changes in the kidney, while 7,8-DHF administration significantly suppressed oxidative stress and NO production. Although the changes in kidney functions with cafeteria diet and 7,8-DHF administration have not been investigated, which is the limiting part of our study, the results of this paper are sufficient to suggest that 7,8-DHF may be protective in obesity-related renal damage. The increasing prevalence of cafeteria diet on eating habits and obesity in the community make these results clinically important. Further studies are needed to examine the renal effects of 7,8-DHF and its effects on other peripheral tissues in more detail.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContribution of the Authors\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors contributed equally to the work.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere is no conflict of interest between the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAebi H. Catalase. Medhods Enzymol. 1984; 105:121-126.\u003c/li\u003e\n \u003cli\u003eAktan F. iNOS-mediated nitric oxide production and its regulation. Life Sci. 2004;75(6):639-653.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAlexakis C, Maxwell P, Bou-Gharios G. Organ-specific collagen expression: implications for renal disease. Nephron Exp Nephrol. 2006;102(3-4):e71-5.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eAykaç G, Uysal M, Yalçın AS, et al. The effect of chronic ethanol ingestion on hepatic lipid peroxide, glutathione, glutathione peroxidase and glutathione transferase in rats. Toxicology. 1985; 36: 71-76.\u003c/li\u003e\n \u003cli\u003eBuege A, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978; 52: 302- 310.\u003c/li\u003e\n \u003cli\u003eBuyukdere Y, Gulec A, Akyol A. Cafeteria diet increased adiposity in comparison to high fat diet in young male rats. PeerJ. 2019;7:e6656.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChan CB, Tse MC, Liu X, Zhang S, Schmidt R, Otten R, Liu L, Ye K. Activation of muscular TrkB by its small molecular agonist 7,8-dihydroxyflavone sex-dependently regulates energy metabolism in diet-induced obese mice. Chem Biol. 2015;22(3):355-368.\u003c/li\u003e\n \u003cli\u003eChoi JW, Lee CW, Lee J, Choi DJ, Sohng JK, Park YI. 7,8-Dihydroxyflavone inhibits adipocyte differentiation via antioxidant activity and induces apoptosis in 3T3-L1 preadipocyte cells. Life Sci. 2016;144:103-112.\u003c/li\u003e\n \u003cli\u003eChoi JW, Lee J, Park YI. 7,8-Dihydroxyflavone attenuates TNF-\u0026alpha;-induced skin aging in Hs68 human dermal fibroblast cells via down-regulation of the MAPKs/Akt signaling pathways. Biomed Pharmacother. 2017;95:1580-1587.\u003c/li\u003e\n \u003cli\u003eCorreia-Costa L, Sousa T, Morato M, Cosme D, Afonso J, Areias JC, Schaefer F, Guerra A, Afonso AC, Azevedo A, Albino-Teixeira A. Oxidative stress and nitric oxide are increased in obese children and correlate with cardiometabolic risk and renal function. Br J Nutr. 2016;116(5):805-815.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDecl\u0026egrave;ves AE, Sharma K. Obesity and kidney disease: differential effects of obesity on adipose tissue and kidney inflammation and fibrosis. Curr Opin Nephrol Hypertens. 2015;24(1):28-36.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eDludla PV, Jack B, Viraragavan A, Pheiffer C, Johnson R, Louw J, Muller CJF. A dose-dependent effect of dimethyl sulfoxide on lipid content, cell viability and oxidative stress in 3T3-L1 adipocytes. Toxicol Rep. 2018;5:1014-1020.\u003c/li\u003e\n \u003cli\u003eEmili M, Guidi S, Uguagliati B, Giacomini A, Bartesaghi R, Stagni F. Treatment with the flavonoid 7,8-Dihydroxyflavone: a promising strategy for a constellation of body and brain disorders. Crit Rev Food Sci Nutr 2020;1-38.\u003c/li\u003e\n \u003cli\u003eF\u0026ouml;rstermann U, Sessa WC. Nitric oxide synthases: regulation and function.Eur Heart J. 2012;33(7):829-37, 837a-837d.\u003c/li\u003e\n \u003cli\u003eGalili O, Versari D, Sattler KJ, Olson ML, Mannheim D, McConnell JP, Chade AR, Lerman LO, Lerman A. Early experimental obesity is associated with coronary endothelial dysfunction and oxidative stress. Am J Physiol Heart Circ Physiol. 2007;292(2):H904-911.\u003c/li\u003e\n \u003cli\u003eG\u0026aacute;mez-M\u0026eacute;ndez AM, Vargas-Robles H, Arellano-Mendoza M, Cruz-Laguna E, Rios A, Escalante B. Early stage of obesity potentiates nitric oxide reduction during the development of renal failure. J Nephrol. 2014;27(3):281-287.\u003c/li\u003e\n \u003cli\u003eGil-Ortega M, Stucchi P, Guzm\u0026aacute;n-Ruiz R, Cano V, Arribas S, Gonz\u0026aacute;lez MC, Ruiz-Gayo M, Fern\u0026aacute;ndez-Alfonso MS, Somoza B. Adaptative nitric oxide overproduction in perivascular adipose tissue during early diet-induced obesity. Endocrinology. 2010;151(7):3299-3306.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGray J, Yeo GS, Cox JJ, Morton J, Adlam AL, Keogh JM, Yanovski JA, El Gharbawy A, Han JC, Tung YC, Hodges JR, Raymond FL, O\u0026apos;rahilly S, Farooqi IS. Hyperphagia, severe obesity, impaired cognitive function, and hyperactivity associated with functional loss of one copy of the brain-derived neurotrophic factor (BDNF) gene. Diabetes. 2006;55(12):3366-3371.\u003c/li\u003e\n \u003cli\u003eGujjala S, Putakala M, Nukala S, Bangeppagari M, Ramaswamy R, Desireddy S. Renoprotective effect of Caralluma fimbriata against high-fat diet-induced oxidative stress in Wistar rats. J Food Drug Anal. 2016;24(3):586-593.\u003c/li\u003e\n \u003cli\u003eHolemans K, Caluwaerts S, Poston L, Van Assche FA. Diet-induced obesity in the rat: a model for gestational diabetes mellitus. Am J Obstet Gynecol. 2004;190(3):858-865.\u003c/li\u003e\n \u003cli\u003eHuai R, Han X, Wang B, Li C, Niu Y, Li R, et al. Vasorelaxing and antihypertensive effects of 7,8-dihydroxyflavone. Am J Hypertens 2014;27(5):750-760.\u003c/li\u003e\n \u003cli\u003eJusto ML, Candiracci M, Dantas AP, de Sotomayor MA, Parrado J, Vila E, Herrera MD, Rodriguez-Rodriguez R. Rice bran enzymatic extract restores endothelial function and vascular contractility in obese rats by reducing vascular inflammation and oxidative stress. J Nutr Biochem. 2013;24(8):1453-1461\u003c/li\u003e\n \u003cli\u003eKanthe PS, Patil BS, Das KK. Terminalia arjuna supplementation ameliorates high fat diet-induced oxidative stress in nephrotoxic rats. J Basic Clin Physiol Pharmacol. 2021; 20200106.\u003c/li\u003e\n \u003cli\u003eKernie SG, Liebl DJ, Parada LF. BDNF regulates eating behavior and locomotor activity in mice. EMBO J. 2000;19(6):1290-1300.\u003c/li\u003e\n \u003cli\u003eKumar D, Dwivedi DK, Lahkar M, Jangra A. Hepatoprotective potential of 7,8-Dihydroxyflavone against alcohol and high-fat diet induced liver toxicity via attenuation of oxido-nitrosative stress and NF-\u0026kappa;B activation. Pharmacol Rep. 2019;71(6):1235-1243.\u003c/li\u003e\n \u003cli\u003eLE Mballa D, Yadang FSA, Tchamgoue AD, Mba JR, Tchokouaha LRY, M Biang E, T Tchinda A, Djomeni Dzeufiet DP, Agbor GA. Cafeteria Diet-Induced Metabolic and Cardiovascular Changes in Rats: The Role of Pişer nigrum Leaf Extract. Evid Based Complement Alternat Med. 2021;2021:5585650.\u003c/li\u003e\n \u003cli\u003eLa Russa D, Giordano F, Marrone A, Parafati M, Janda E, Pellegrino D. Oxidative Imbalance and Kidney Damage in Cafeteria Diet-Induced Rat Model of Metabolic Syndrome: Effect of Bergamot Polyphenolic Fraction. Antioxidants (Basel). 2019;8(3):66\u003c/li\u003e\n \u003cli\u003eLakkis JI, Weir MR. Obesity and Kidney Disease. Prog Cardiovasc Dis. 2018;61(2):157-167.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eLee W, Eom DW, Jung Y, Yamabe N, Lee S, Jeon Y, Hwang YR, Lee JH, Kim YK, Kang KS, Kim SN. Dendrobium moniliforme attenuates high-fat diet-induced renal damage in mice through the regulation of lipid-induced oxidative stress. Am J Chin Med. 2012;40(6):1217-1228.\u003c/li\u003e\n \u003cli\u003eLeffa DD, da Silva J, Daumann F, Dajori AL, Longaretti LM, Damiani AP, de Lira F, Campos F, Ferraz Ade B, C\u0026ocirc;rrea DS, de Andrade VM. Corrective effects of acerola (Malpighia emarginata DC.) juice intake on biochemical and genotoxical parameters in mice fed on a high-fat diet. Mutat Res. 2014;770:144-152.\u003c/li\u003e\n \u003cli\u003eManna P, Jain SK. Obesity, oxidative stress, adipose tissue dysfunction, and the associated health risks: causes and therapeutic strategies. Metab Syndr Relat Disord. 2015;13(10):423-444.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMartin B, Caron N, Jadot I, Colombaro V, Federici G, Depommier C, Decl\u0026egrave;ves A\u0026Eacute;. Evaluation of inducible nitric oxide synthase inhibition on kidney function and structure in high-fat diet-induced kidney disease. Exp Physiol. 2018;103(1):125-140.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eMiranda KM, Espey MG, Wink DA. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001;5(1):62-71.\u003c/li\u003e\n \u003cli\u003eMorais Mewes J, Rodrigues Silva Gasparin F, Yoshida T, Am\u0026acirc;ncio Daniel da Silva M, Raquel Mar\u0026ccedil;al Natali M, Francisco Veiga Bizerra P, Sayuri Utsunomiya K, Hideo Gilglioni E, Shigueaki Mito M, Cristiane Mantovanelli G, Thais Lima de Souza B, Makiyama Klosowski E, Luiza Ishii-Iwamoto E, Constantin J, Polimeni Constantin R.: The Role of Mitochondria in Sex-Dependent Differences in Hepatic Steatosis and Oxidative Stress in Response to Cafeteria Diet-Induced Obesity in Mice. Nutrients. 2019;11(7):1618.\u003c/li\u003e\n \u003cli\u003eNoeman SA, Hamooda HE, Baalash AA. Biochemical study of oxidative stress markers in the liver, kidney and heart of high fat diet induced obesity in rats. Diabetol Metab Syndr. 2011;3(1):17.\u003c/li\u003e\n \u003cli\u003eNoronha BT, Li JM, Wheatcroft SB, Shah AM, Kearney MT. Inducible nitric oxide synthase has divergent effects on vascular and metabolic function in obesity. Diabetes. 2005;54(4):1082-1089.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eOzata M, Mergen M, Oktenli C, Aydin A, Sanisoglu SY, Bolu E, Yilmaz MI, Sayal A, Isimer A, Ozdemir IC. Increased oxidative stress and hypozincemia in male obesity. Clin Biochem. 2002;35(8):627-631.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePandey SN, Kwatra M, Dwivedi DK, Choubey P, Lahkar M, Jangra A. 7,8-Dihydroxyflavone alleviated the high-fat diet and alcohol-induced memory impairment: behavioral, biochemical and molecular evidence. Psychopharmacology (Berl). 2020;237(6):1827-1840.\u003c/li\u003e\n \u003cli\u003ePark HY, Park C, Hwang HJ, Kim BW, Kim GY, Kim CM, et al. 7,8-Dihydroxyflavone attenuates the release of pro-inflammatory mediators and cytokines in lipopolysaccharide-stimulated BV2 microglial cells through the suppression of the NF-\u0026kappa;B and MAPK signaling pathways. Int J Mol Med 2014;33(4):1027-1034.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePellegrino D, La Russa D, Marrone A. Oxidative imbalance and kidney damage: new study perspectives from animal models to hospitalized patients. Antioxidants (Basel). 2019;8(12):594.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePelleymounter MA, Cullen MJ, Wellman CL. Characteristics of BDNF-induced weight loss. Exp Neurol. 1995;131(2):229-238.\u003c/li\u003e\n \u003cli\u003ePinheiro IRR, Melo MFN, de Sousa SV, Cardoso BG, da Silva TM, Rangel LP, Cortes VF, de Lima Santos H, Chaves VE, Garcia IJP, Barbosa LA. Evaluation of the effect of cafeteria diet on the kidney Na,K-ATPase activity, and oxidative stress. J Cell Biochem. 2019;120(11):19052-19063.\u003c/li\u003e\n \u003cli\u003ePommer W. Preventive nephrology: the role of obesity in different stages of chronic kidney disease. Kidney Dis (Basel). 2018;4(4):199-204.\u003c/li\u003e\n \u003cli\u003eSharma K. Obesity and diabetic kidney disease: role of oxidant stress and redox balance. Antioxid Redox Signal. 2016; 25(4): 208\u0026ndash;216.\u003c/li\u003e\n \u003cli\u003eSun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem. 1988; 34: 497-500.\u003c/li\u003e\n \u003cli\u003eTang J, Yan H, Zhuang S. Inflammation and oxidative stress in obesity-related glomerulopathy. Int J Nephrol. 2012;2012:608397.\u003c/li\u003e\n \u003cli\u003eTsch\u0026ouml;p M, Heiman ML. Rodent obesity models: an overview. Exp Clin Endocrinol Diabetes. 2001;109(6):307-319.\u003c/li\u003e\n \u003cli\u003eTsuchiya K, Sakai H, Suzuki N, Iwashima F, Yoshimoto T, Shichiri M, Hirata Y. Chronic blockade of nitric oxide synthesis reduces adiposity and improves insulin resistance in high fat-induced obese mice. Endocrinology. 2007;148(10):4548-4556.\u003c/li\u003e\n \u003cli\u003eTsuda K, Nakatani T, Sugama J, Okuwa M, Sanada H. Influence of the timing of switching a protein-free to a protein-containing diet on the wound healing process in a rat all-layer skin defect. Int Wound J. 2010; 7(3), 135-146.\u003c/li\u003e\n \u003cli\u003eWang B, Wu N, Liang F, Zhang S, Ni W, Cao Y, et al. 7,8-dihydroxyflavone, a small-molecule tropomyosin-related kinase B (TrkB) agonist, attenuates cerebral ischemia and reperfusion injury in rats. J Mol Histol 2014;45(2):129-140.\u003c/li\u003e\n \u003cli\u003eWang Y, Chen X, Song Y, Caballero B, Cheskin LJ. Association between obesity and kidney disease: a systematic review and meta-analysis. Kidney Int. 2008;73(1):19-33.\u003c/li\u003e\n \u003cli\u003eWolf G, Ziyadeh FN. Leptin and renal fibrosis. Contrib Nephrol. 2006;151:175-183.\u003c/li\u003e\n \u003cli\u003eWood J, Tse MCL, Yang X, Brobst D, Liu Z, Pang BPS, Chan WS, Zaw AM, Chow BKC, Ye K, Lee CW, Chan CB. BDNF mimetic alleviates body weight gain in obese mice by enhancing mitochondrial biogenesis in skeletal muscle. Metabolism. 2018;87:113-122.\u003c/li\u003e\n \u003cli\u003eYeo GS, Connie Hung CC, Rochford J, Keogh J, Gray J, Sivaramakrishnan S, O\u0026apos;Rahilly S, Farooqi IS. A de novo mutation affecting human TrkB associated with severe obesity and developmental delay. Nat Neurosci. 2004;7(11):1187-1189.\u003c/li\u003e\n \u003cli\u003eZeeni N, Dagher-Hamalian C, Dimassi H, Faour WH. Cafeteria diet-fed mice is a pertinent model of obesity-induced organ damage: a potential role of inflammation. Inflamm Res. 2015;64(7):501-512.\u003c/li\u003e\n \u003cli\u003eZeng Y, Lv F, Li L, Yu H, Dong M, Fu Q. 7,8-dihydroxyflavone rescues spatial memory and synaptic plasticity in cognitively impaired aged rats. J Neurochem. 2012;122(4):800-811.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhao P, Li X, Li Y, Zhu J, Sun Y, Hong J. Mechanism of miR-365 in regulating BDNF-TrkB signal axis of HFD/STZ induced diabetic nephropathy fibrosis and renal function. Int Urol Nephrol. 2021;53(10):2177-2187.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cafeteria diet, obesity-induced renal damage, oxidative stress, 7,8-DHF, nitric oxide","lastPublishedDoi":"10.21203/rs.3.rs-2053626/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2053626/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eIn this study, the possible protective effect of 7,8-Dihydroxyflavone (7,8-DHF), a brain-derived neurotrophic factor (BDNF) mimetic and anti-oxidant flavonoid, in renal damage caused by cafeteria diet-induced obesity was investigated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod: \u003c/strong\u003eIn the study, 4-5 week old C57BL/6 male mice were used and the subjects were divided into 4 groups as Control, CD (cafeteria diet), CD+Vehicle and CD+7,8-DHF (n=9-11). Control group subjects were fed with chow diet for 16 weeks and other groups were fed with cafeteria diet. In the last 28 days of the feeding period, 7,8-DHF treatment (5 mg/kg/day, intraperitoneal) was administered in the CD+7,8-DHF group, and DMSO (17%) as a 7,8-DHF carrier was administered in the CD+Vehicle group. At the end of 16 weeks, the subjects were sacrificed and malondialdehyde (MDA), reduced glutathione (GSH), nitrite + nitrate (NOx) and collagen levels, and superoxide dismutase (SOD) and catalase (CAT) enzyme activities were measured in kidney tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e At the end of 16 weeks, body weights of all subjects increased compared to baseline. Weight gain was higher in CD (p\u0026lt;0.001) and CD+Vehicle groups (p\u0026lt;0.001) compared to control. The weight gain in the CD+7,8-DHF group was not different from the control. Compared to the CD group, the weight gains in the CD+Vehicle and CD+7,8-DHF groups were lower. Compared to the control group CD group had higher renal MDA level (p\u0026lt;0.0001), lower GSH level (p\u0026lt;0.0001), less SOD (p\u0026lt;0.0001) and CAT (p\u0026lt;0.0001) activity, lower NOx (p\u0026lt;0.0001) and collagen (p\u0026lt;0.0001) levels. Vehicle administration did not affect these parameters as results were similar to CD group. However, significant changes were noted with 7,8-DHF treatment. Compared with the CD+Vehicle group, the CD+7,8-DHF group had lower MDA levels (p\u0026lt;0.001), higher GSH levels (p\u0026lt;0.001), lower NOx levels (p\u0026lt;0.001), higher SOD (p\u0026lt;0.001) and CAT (p\u0026lt;0.001) activities. 7,8-DHF treatment brought these parameters closer to the control values, but did not fully return to control, except for CAT activity. Renal collagen deposition was not affected by 7,8-DHF treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Oxidative stress plays an important role in obesity-induced renal damage. 7,8-DHF may be important in the suppression of renal damage in cafeteria diet-induced obesity, at least by inhibiting oxidative stress and excessive nitric oxide production. The increasing prevalence of eating habits and obesity together with the cafeteria diet in the society, makes these results clinically important. These effects of 7,8-DHF need to be investigated in more details.\u003c/p\u003e","manuscriptTitle":"Renal effects of 7,8-Dihydroxyflavone in cafeteria diet-induced obesity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-09-14 20:49:25","doi":"10.21203/rs.3.rs-2053626/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9b0dd31a-1e99-44d6-b514-2e93aa491e63","owner":[],"postedDate":"September 14th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2022-09-15T18:59:21+00:00","versionOfRecord":[],"versionCreatedAt":"2022-09-14 20:49:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2053626","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2053626","identity":"rs-2053626","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}