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.