The effect of tropisetron on oxidative stress, SIRT1, FOXO3a, and claudin-1 in the renal tissue of STZ-induced diabetic rats

Abstract

Tropisetron is a 5-HT3 receptor antagonist that exerts protective effect against DN. The aim of this study was to investigate the possible molecular mechanisms associated with the renoprotective effects of tropisetron in STZ-induced diabetic rats. Animals were subdivided into 5 equal groups; control, tropisetron, diabetes, tropisetron + diabetes, and glibenclamide + diabetes (n = 7). For induction of type 1 diabetes, a single injection of STZ (55 mg/kg, i.p.) was administered to the animals. Diabetic rats were treated with tropisetron (3 mg/kg) and glibenclamide (1 mg/kg) for 2 weeks. According to the conducted analysis, diabetes led to renal dysfunction (reduction in glomerular filtration rate and urine urea and creatinine as well as elevation in plasma urea and creatinine) and abnormalities in antioxidant defense system (reduction in TAC and elevation in MDA), compared with the control group, which was prevented by tropisetron treatment. Reverse transcription–quantitative polymerase chain reaction and western blotting analysis demonstrated that SIRT1 gene expression decreased while FOXO3a and NF-κB gene expression as well as phosphorylated FOXO3a/total FOXO3a protein ratios and claudin-1 protein level increased in the kidney of diabetic rats compared with the control group. Herein, the results of this research showed that tropisetron treatment reversed these changes. Besides, all these changes were comparable with those produced by glibenclamide as a positive control. Hence, tropisetron ameliorated renal damage due to diabetic nephropathy possibly by suppressing oxidative stress and alteration of SIRT1, FOXO3a, and claudin-1 levels.

Electronic supplementary material

The online version of this article (10.1007/s12192-020-01170-5) contains supplementary material, which is available to authorized users.

Introduction

Diabetic nephropathy (DN) is a micro-vascular disease which is characterized by excessive proteinuria with the morbidity of 25–40% (Chen et al. 2011; Ritz et al. 2010). Morphological and functional alterations including glomerular hypertrophy, excessive accumulation of extracellular matrix and glomerulosclerosis, and, ultimately, loss of renal function in the kidney are associated with hyperglycemia and, subsequently, glucose metabolism disorder (Kanwar et al. 2008; Vikram et al. 2014). Therefore, special attention has been given to effective management or treatment which may be necessary for combating the disease at the early stage. Accumulating evidence indicates that production of reactive oxygen species (ROS) as a direct issues of hyperglycaemia and subsequently, production of inflammatory cytokines are major mechanisms in the development of diabetic complications (Beckman and Ames 1998; Giacco and Brownlee 2010; Jaimes et al. 2010; Kiritoshi et al. 2003). It is evident that imbalance between pro-oxidant and antioxidant defense capacity results in free radical accumulation which may damage cellular macromolecules including DNA as well as protein modification and lipid peroxidation (Peluso and Raguzzini 2016).

The mammalian sirtuins are a family of NAD+-dependent deacetylases, including seven members (SIRT1-7). Among them, SIRT1, the first family member, has most extensively been studied (Dong et al. 2014). Recent studies have revealed that SIRT1 plays an important role in the regulation of cell death/longevity as well as acute and chronic stress response in mammals by attenuating DNA damage, inflammation, and apoptosis (Hasegawa et al. 2013; Yun et al. 2012). Several studies point out higher levels of ROS-induced downregulation of SIRT1 activity in diabetic kidney, both in vivo and in vitro (Akhtar and Siragy 2019; Du et al. 2016; Kumar et al. 2014; Papadimitriou et al. 2015; Park et al. 2016; Shi and Huang 2018).

SIRT1 can exert its action by regulating several proteins, such as the transcription factors of nuclear factor-kappaB (NF-κB) and FOXO family (Brunet et al. 2004; Langley et al. 2002; Motta et al. 2004; Yeung et al. 2004). Recently, it has been notified that SIRT1 regulates glucose metabolism by contributing to FOXOs (Kobayashi et al. 2005). FOXO3a is a subgroup of the FOXO family of transcription factors which have an important role during oxidative stress (Accili and Arden 2004). Modifications of FOXO3a occur by phosphorylation and acetylation which can appear simultaneously in various conditions. According to the literature, SIRT1 can deacetylate FOXO3a, thus increasing its ubiquitination level and protecting against stressors (Wang et al. 2017a). There is little information about the mechanism of the interactions between SIRT1 and FOXO during hyperglycemic conditions (Yun et al. 2012). However, it was suggested that SIRT1 modulate the activity of the FOXO probably by their nuclear translocation (Brunet et al. 2004) and also controls the gene-specific transcription (Kobayashi et al. 2005). Therefore, translocation of FOXO3a from the cytoplasm to the nucleus is mainly caused by deacetylation activity of SIRT1, especially in oxidative stress status (Yun et al. 2012).

Wang et al. reported that high glucose treatment modulated SIRT1 and FOXO3a protein expression in human kidney epithelial cells (Wang et al. 2017b). Moreover, FOXO3a functions as a positive regulator of NF-κB signaling, in which it could be employed to affect cell survival strategies under stress conditions (Li et al. 2012).

Claudin-1 is a putative parietal epithelial cell (PEC)-specific marker (Huby et al. 2009; Ohse et al. 2009; Rincon-Choles et al. 2006), which is negatively associated with SIRT1 expression in both proximal tubules and glomerular region. It was shown that SIRT1 downregulation in diabetes is corroborated by the upregulation of the tight junction protein claudin-1 in podocytes contributing to albuminuria (Gong et al. 2017; Hasegawa et al. 2013). Since oxidative stress, inflammatory cytokine, and their molecular mediators are major topics in the deleterious effect of diabetes, antioxidant and anti-inflammatory agents could provide novel therapies against DN (Elmarakby and Sullivan 2012).

Tropisetron is a recently emerging 5-HT3 receptor antagonist which has been identified as an antiemetic drug during chemotherapy (Barzegar-Fallah et al. 2015). Several reports have shown that tropisetron can offer antiapoptotic, antidiabetic, anticancer, antilipidemic, neuroprotective, and cardioprotective effects which are probably mediated through antioxidant and anti-inflammatory mechanisms (Aminzadeh 2017; Asadi et al. 2016; Barzegar-Fallah et al. 2015; Barzegar-Fallah et al. 2014; Gholizadeh-Ghaleh Aziz et al. 2019; Rashidi and Bazi 2020). There are some studies that prove anti-oxidative effect of tropisetron, both in vitro and in vivo, such as protective effect of tropisetron on hyperglycemia-induced oxidative disturbance in PC12 cells (Aminzadeh 2017), amelioration of the mitochondrial injury by lowering nitrergic system’s activity in the brain (Haj-Mirzaian et al. 2016), or attenuating oxidative stress against brain aging in mice (Mirshafa et al. 2020).

Recently, Barzegar Fallah et al. revealed that tropisetron ameliorates blood glucose and also renal function as evidenced by decreasing serum blood urea nitrogen (BUN), serum creatinine levels, and urinary albumin excretion.. In addition, malondialdehyde (MDA) content, glutathione (GSH) levels, superoxide dismutase (SOD), and catalase (CAT) activities were improved in diabetic animals treated with tropisetron (Barzegar-Fallah et al. 2015). However, there is no other strong evidence about the modulation of DN by tropisetron and its molecular mechanism. This study, therefore, aims to evaluate, first: the effects of tropisetron on molecular mediators including SIRT1, NF-κB, and FOXO3a, as well as claudin-1, which is a protein involved in tight epithelial junctions and plays a major role in renal diabetic nephropathy; second, to compare the effect of tropisetron with the standard marketed antidiabetic drug, glibenclamide, in order to find a more beneficial and safe agent as a gold standard antidiabetic drug.

Materials and methods

This study was conducted according to the Principles of Laboratory Animal Care in Urmia University of Medical Sciences. Ethical approval was achieved from the EU Directive 2010/63/EU for animal experiments according to the guidelines of the Medical Ethics Committee, Ministry of Health, Iran (IR.UMSU.REC.1397.291). Thirty-five male Wistar rats (230–270 g weight, 3–4 months age) were allocated into 5 groups (seven rats in each group):

1. Control group: Rats received normal saline intraperitoneally for 2 weeks.

2. Tropisetron group: Rats received tropisetron intraperitoneally for 2 weeks.

3. Diabetes group: Diabetes was induced by streptozotocin (STZ) injection in rats.

4. Tropisetron + diabetes group: The rats received 3 mg/kg of tropisetron (Gholizadeh-Ghaleh Aziz et al. 2019) intraperitoneally for 2 weeks after diabetes induction.

5. Glibenclamide + diabetes group: The rats received 1 mg/kg glibenclamide (Gholizadeh-Ghaleh Aziz et al. 2019) intraperitoneally for 2 weeks after diabetes induction.

Induction of diabetes

Streptozotocin (50 mg/kg) was intraperitoneally injected in a single dose to induce diabetes in rats. Based on this method, type I diabetes was induced in the rats 72 h after injection. Diabetes was diagnosed and confirmed by creating a minor injury using a lancet in the tail of fasting rats, and then a drop of blood was placed on a glucometer strip. Then, it was measured by a glucometer (Boehringer Mannheim Indianapolis, IN), and blood glucose levels above 300 mg/dl were considered an indicator of diabetes induction.

After 2 weeks and 24 h before anesthetizing, rats were placed in metabolic cages one by one, and urine was collected. Then, urine samples were centrifuged right away with transparent surface of samples stored at − 20 °C for analysis of urea and creatinine. Afterwards, the rats were weighted and anesthetized by intraperitoneal injection of ketamine (60 mg/kg) and xylazine (4 mg/kg) mixture. Then, the abdominal cavity was opened; the blood obtained from heart by a narrow syringe and mixed with ethylenediaminetetraacetic acid (EDTA) and centrifuged at 4000×g for 20 min. Then, the yielded plasma was stored at − 80 °C for later measurement of urea, creatinine, and total antioxidant capacity (TAC). Both kidneys of the animals were excised, washed with cold physiological serum, and weighted. The left kidney tissues were hemogenized by Ultra Turrax (T10B, IKA, and Germany) in an RNAase-containing solution to test the expression levels of SIRT1, NF-κB, and FOXO3a using the real-time-PCR method. Right kidney was frozen at − 80 °C for MDA measurement and western blot analysis (total and phosphorylated FOXO3a and claudin-1).

Fasting blood glucose

At the end of protocol, after overnight fasting (14 to18 h), blood samples were obtained from the tip of the tail, and then glucose levels were measured by using a digital glucometer (Elegance, CT-X10, Frankenberg, Germany).

Urinary and plasma urea and creatinine

For this purpose, the urinary and plasma urea and creatinine levels will be measured by economic kits specific for measurement of urea and creatinine (Biotechnical; Varginha, Minas Gerais, Brazil).

Glomerular filtration rate

Glomerular filtration rate (GFR) is a best overall index of kidney functions, which was evaluated by calculating creatinine clearance (GFR = [UCr × V]/SCr) and using plasma and urine creatinine concentrations and the urine flow rate or volume.

Malondialdehyde and total antioxidant capacity

MDA as the end produce of lipid peroxidation was assessed through a reaction with thiobarbituric acid (Sigma-Aldrich; USA) in the kidney samples according to manufacturer’s protocol (Esterbauer and Cheeseman 1990). In brief, 0.3–0.4 g of the kidney tissue was homogenized in ice-cold KCl (150 mM) and then centrifuged at 3000×g for 10 min. Then, 0.5 ml of the supernatant was combined with 3 ml phosphoric acid (1%, v/v), and following vortex mixing, 2 ml of 6.7 g/l TBA was subjoined to the samples. The specimens were heated at 100 °C for 45 min. After cooling down on ice, n-butanol (3 ml) was combined and the products were further centrifuged at 3000×g for another 10 min. Then, the absorbance of the products was considered at 532 nm through spectrophotometry. The absorbance level was compared with the standard curve. TAC was measured by a Randox total antioxidant status kit, in which ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfanate) is incubated with peroxidase and H₂O₂ resulting in the radical cation ABTS + production. This has a stable blue-green color, which is evaluated using a 600-nm automatic analyzer (Amini et al. 2020).

Primer design and real-time PCR

The RNA was first extracted by the relevant kit (GENALL), and the condensed RNA was qualitatively (electrophoresis) and quantitatively (nano-drop) assessed in order to ensure RNA extraction and application in subsequent molecular techniques. Then, the relevant cDNA was synthesized from all extracted RNA samples. The primers of the target genes and the glyceraldehyde dehydrogenase (GAPDH) gene as a house-keeping gene were examined on the NCBI site using the Generunner software (Supplementary Table). Then, gene expression was analyzed using the synthesized primers by a PCR kit, followed by statistical analysis of data.

Western blot

Western blot method was used to determine FOXO3a (total and phosphorylated) and claudin-1 protein level in the kidney. Two resolving and stacking gels are used to make the electrophoresis gel. After preparing the resolving gel and pouring it into the mold, a stacking gel was added after an hour and after stacking. The samples were loaded into wells made by a special comb. Then they were separated by SDS-PAGE. Within an hour, all the loaded specimens were transferred to the PVDF membrane. Subsequently, the specimens were blocked with 5% skim milk buffer including 0.1% Tween 20 and were then incubated with primary antibodies overnight at 4 °C in a shaker incubator. Then the membranes were incubated with horseradish peroxidase-conjugated secondary antibody. Finally, beta-actin was used as an internal standard for the western blot analysis. At the end, the effects of blotted samples, depending on the level of target protein, were evaluated on the radiography film in a dark room. To quantify the target proteins, the film scan results were calculated by a densitometer relative to the level of actin. The antibodies used in Western blotting assays are shown in the Supplementary Table (catalog numbers and companies).

Statistical analysis

Results were presented as mean ± SEM. Statistical analysis was carried out using SPSS 16.0. Comparison between groups was estimated by ANOVA test followed by the Tukey’s post hoc test. The differences were assumed statistically significant at P < 0.05.

Results

Tropisetron decreased fasting blood glucose (mg/dl), kidney weight (g), and kidney index (mg/g), while increased body weight (g) in the STZ-induced diabetic rats

Similarly to previous studies in this work, diabetic groups showed severe (P < 0.001) hyperglycemia compared with the control group. Tropisetron (P < 0.01) and glibenclamide (P < 0.05) consumption for 2 weeks significantly reduced blood glucose in STZ-induced diabetic groups, but it was still significantly higher than that in the control group (P < 0.001). There was no significant difference in body weight (BW) among the groups at the beginning of this study (data not shown). Induction of diabetes by STZ significantly decreased (P < 0.001) finally (BW) in diabetic rats. Kidney weight (KW) was not significant among the groups at first; however, it was increased in diabetic group and decreased after interventions. Kidney index (KW/BW) was significantly increased (P < 0.001) in T1DM when compared with the control group. Interestingly, after 2 weeks administration of tropisetron and glibenclamide, modulated KW/BW (P < 0.05) (Table 1).

Table 1.

Effect of tropisetron treatment on diabetes-induced changes in FBS, BW, KW, and KI in the different groups of rats at the end of experiment

Groups	Control	Tropisetron	Diabetes	Tropisetron + diabetes	Glibenclamide + diabetes
FBS (mg/dl)	86 ± 7.1	91 ± 12.8	564 ± 35.6***	313 ± 43.7***,$$	356 ± 78.8***,$
BW (g)	292 ± 5.9	276 ± 7.7	175 ± 7.2***	213 ± 11.7$***	227 ± ±9.7$$***
KW (mg)	1010 ± 40.3	865 ± 47.8	1198 ± 101	998 ± 143	1138 ± 73
KI (mg/g)	3.44 ± 0.1	3.13 ± 0.16	6.83 ± 0.44***	4.78 ± 0.81$	5 ± 0.31$

FBS, fasting blood glucose; BW, body weight; KW, kidney weight; KI, kidney index

All data are expressed as mean ± SEM (n=7): *** P < 0.001 vs control group. $ P < 0.05, $$ P < 0.01 vs diabetic group

Tropisetron improved biochemical analysis in the STZ-induced diabetic rats

As in previous studies, urea levels in the plasma and urine of the diabetic group were significantly higher than those in the control group (P < 0.001). In the tropisetron + diabetes group, plasma (P < 0.001) and urine (P < 0.05) urea levels decreased significantly compared with those in the diabetes groups. However, urine urea was still significantly lower than that in the control group (P < 0.01). There is no significant difference in plasma urea level between tropisetron + diabetes and control groups. The plasma (P < 0.001) and urine (P < 0.05) creatinine level sowed a significant increase in the diabetic rats compared with that in the control group. Tropisetron administration reduced the urine and plasma creatinine levels significantly (P < 0.001) compared with diabetic group. In diabetic animals, the creatinine clearance, as an indicator of glomerular filtration rate, was significantly lower than that in the control group (P < 0.05). Tropisetron treatment significantly increased the creatinine clearance compared with that in the diabetes group (P < 0.001). The same results were obtained in the glibenclamide-treated group. However, the plasma urea is much lower in the tropisetron-treated group compared with that in the glibenclamide + diabetes group (P < 0.01) (Table 2).

Table 2.

Effect of tropisetron treatment on diabetes-induced changes in kidney, urine, and plasma levels of creatinine, urea, and creatinine clearance in the different groups of rats at the end of experiment

Groups	Control	Tropisetron	Diabetes	Tropisetron + diabetes	Glibenclamide + diabetes
Urine urea (mg/dl)	188 ± 12.32	176.16 ± 11.48	87.66 ± 8.69***	127.33 ± 6$,**	126.66 ± 8.97$,**
Plasma urea (mg/dl)	79.83 ± 3.24	81.66 ± 3.35	121.33 ± 3.74***	89 ± 4.3$$$	107.66 ± 1.64$,***,&&
Urine creatinine (mg/dl)	7.46 ± 0.17	8.88 ± 0.77	3.62 ± 0.47*	10.03 ± 1.28$$$	10.5 ± 0.98$$$
Plasma creatinine (mg/dl)	0.6 ± 0.03	0.98 ± 0.17	1.88 ± 0.04***	0.95 ± 0.06$$$	1.55 ± 0.02$
Creatinine clearance (ml/min)	0.08 ± 0.01	0.06 ± 0.01	0.02 ± 0.003*	0.13 ± 0.02$$$	0.08 ± 0.008$

All data are expressed as mean ± SEM (n = 7)

*P < 0.05, **P < 0.01, and ***P < 0.001 vs. control group; ^$P < 0.05 and ^$$$P < 0.001 vs. diabetic group; ^&&P < 0.01 vs. tropisetron + diabetes group

Tropisetron increased plasma TAC content and attenuated MDA level in the STZ-induced diabetic rats

MDA level in control, tropisetron, diabetes mellitus, tropisetron +diabetes, and glibenclamide + diabetes groups was 8.65 ± 1.01, 10.89 ± 1.6 ، 35.54 ± 7.1, 18.76 ± 3.6, and 16.89 ± 4.1 nmol/mg of protein, respectively. Our study, along with those of others, have demonstrated the highest level of MDA in the diabetes group, which showed a significant increase compared with the control group (P < 0.001). MDA level in the tropisetron + diabetes and glibenclamide + diabetes groups showed a significant reduction (P < 0.01) compared with the diabetes group (Fig. 1a). TAC levels in the control, tropisetron, diabetes, tropisetron + diabetes, and glibenclamide + diabetes groups were 0.65 ± 0.03, 0.76 ± 0.06, 0.26 ± 0.08, 0.45 ± 0.06, and 0.57 ± 0.08 nmol/ml, respectively. TAC levels decreased in the diabetes group compared with that of the control group (P < 0.001), as shown previously. Both tropisetron and glibenclamide administrations significantly (P < 0.01) increased plasma TAC levels in diabetic animals in comparison with the diabetic group (Fig. 1b).

Fig. 1.

Effect of tropisetron treatment on the renal MDA (a) and TAC (b) levels in the different groups. *P < 0.05 and ***P < 0.001 vs. control group; ^$$P < 0.01 vs. diabetes group. All data are expressed as the means ± SEM (n = 7)

Tropisetron increased SIRT1 and decreased FOXO3a and NF-κB gene expressions

Gene expressions were evaluated by real-time PCR which provided important evidence for a difference between groups. Accordingly, diabetes was associated with a significant decrease (0.65 ± 0.08) in SIRT1 gene expression (P < 0.01) and a significant increase (P < 0.001) in FOXO3a (1.98 ± 0.08) and NF-κB (1.71 ± 0.14) gene expression compared with the control group. Tropisetron treatment increased SIRT1 (0.88 ± 0.3) and decreased FOXO3a (1.53 ± 0.08) (P < 0.05) and NF-κB (1.27 ± 0.03) (P < 0.01) mRNA levels significantly in diabetic rats (Fig. 2a–c). Similar results were provided in the animals treated with the standard drug, glibenclamide.

Fig. 2.

Effect of tropisetron treatment on the renal SIRT1 (a), FOXO3a (b), and NF-κB (c) gene expressions in different groups. **P < 0.01 and ***P < 0.001 vs. control group; ^$P < 0.05 and ^$$P < 0.01 vs. diabetic group. All data are expressed as the means ± SEM (n = 7)

Tropisetron decreased FOXO3a and claudin-1 protein expressions

To evaluate FOXO3a (total and phosphorylated) and claudin-1 protein expression following tropisetron and glibenclamide treatment, western blotting was used in the kidney of different groups. The data analysis exhibit a significant increase in phosphorylated FOXO3a/total FOXO3a protein ratios (4.6 ± 0.17) and claudin-1 (3.02 ± 0.16) protein levels in the diabetic group compared with the control (P < 0.001). Administration of tropisetron markedly decreased phosphorylated FOXO3a/total FOXO3a protein ratios (2.14 ± 0.31) and claudin-1 (2.4 ± 0.12) levels in the kidney of diabetic rats (P < 0.01). Glibenclamide treatment significantly attenuated both phosphorylated FOXO3a/total FOXO3a protein ratios (2.57 ± 0.5) (P < 0.001) and claudin-1 (2.2 ± 0.11) (P < 0.05) protein levels in the STZ-induced diabetic rats (Fig. 3a–c).

Fig. 3.

Effect of tropisetron treatment on the renal protein levels. The blotting images of FOXO3a (total and phosphorylated) and claudin-1 (a). The bar charts represent the quantitative analysis of phosphorylated FOXO3a/total FOXO3a protein ratios (b) and claudin-1 (c) normalized against β-actin. *P < 0.05 and ***P < 0.001 vs. control group; ^$P < 0.05 and ^$$P < 0.01 vs. diabetic group. a control; b tropisetron; c diabetes; d tropisetron + diabetes; e glibenclamide + diabetes. All data are expressed as the means ± SEM (n = 7)

Discussion

The findings of the current study are given in the following sections: single injection of STZ-induced T1DM was associated with severe hyperglycemia, weight loss, and kidney dysfunction, which is manifested by a marked increase of plasma creatinine and urea and a marked decrease in creatinine clearance as an index of GFR compared with that in the control group. STZ disrupted the oxidative balance in the kidney tissue that was proved by increasing MDA and decreasing the TAC content. Further molecular analysis showed that diabetes decreased SIRT1 mRNA and increased NF-κB mRNA as well as phosphorylated FOXO3a/total FOXO3a protein ratios and claudin-1 protein level in the kidney of male rats. Significant amelioration of urea, creatinine, creatinine clearance, as well as SIRT1 mRNA, NF-κB mRNA, phosphorylated FOXO3a/total FOXO3a protein ratios, and claudin-1 protein, similar to those of the control animals, were observed in the tropisetron + diabetes group.

DN which is characterized by several features such as disruption in renal structural and functional integrity, increase in kidney size, as well as glomerular and tubular hypertrophy, is a leading cause of end-stage renal disease (Singh et al. 2018; Yaribeygi et al. 2018). Progressive albuminuria, decreased GFR, and arterial blood pressure elevation are the most pronounced functional symptoms observed in diabetic patients (Alzahrani et al. 2020). The earliest marker of DN is albuminuria which is caused due to a progressive glomerular damage as a central feature in initiating kidney injury (Elsherbiny et al. 2018). Consistent with our findings, previous studies highlighted that DN is accompanied by increments in serum creatinine and urea suggesting dysfunction of the renal tissue (Sharma et al. 2006; Tang et al. 2018).

Accumulating evidence indicates that generation of free radicals due to hyperglycemia is a key event in the onset and progression of diabetic microvascular complications including DN. Excessive ROS production contributes to oxidative damage by affecting macromolecules, cells, and tissues and results in irreversible insult or functional disturbances (Kiritoshi et al. 2003).

MDA as a marker of lipid peroxidation is triggered by the attack of free radicals on membrane unsaturated fatty acids, which have contributed to diabetic complications (Pieme et al. 2017). Total antioxidant status was estimated by TAC which is a biomarker to determine oxidative stress in many pathological conditions. Thus, MDA and TAC impairments in diabetic patients reflect the production of free radicals which induce oxidative stress status (Peluso and Raguzzini 2016). Consistently, the results of this study showed increased MDA and decreased TAC levels in diabetic rat kidney. Considering this fact, attenuation of oxidative stress ameliorated diabetic complications including renal injury (Alzahrani et al. 2020; Barzegar-Fallah et al. 2015).

Tropiserton, as a 5-HT3 antagonist with antioxidant and anti-inflammatory activities, shows protective effects in several pathological conditions such as diabetes (Barzegar-Fallah et al. 2015; Rashidi and Bazi 2020). The nephroprotective effect of tropisetron was previously described in animal experiments. Within this view, tropisetron ameliorated DN in a 5-HT3 receptor-independent manner by preventing increased blood glucose, renal MDA, CAT, SOD, Gpx, and TNF-α levels and decreased urinary cytokine excretion and albuminuria (Barzegar-Fallah et al. 2015).

In line with this issue, in the current investigation, the evidence is provided to show that tropisetron modulated hyperglycemia, weight loss, and oxidative stress status which is exhibited by decreasing MDA and increasing the TAC content and improved kidney dysfunction (a significant depletion in plasma urea and creatinine). Glibenclamide was applied as a standard drug according to previous studies (Amini et al. 2020). Interestingly, no significant difference was observed in the mentioned parameters in glibenclamide-administered and tropisetron-administered groups in the kidney of diabetic rats. However, the precise mechanisms behind the protective effect of tropisetron in DN are still unknown. To the best of our knowledge, this is the first study to demonstrate that tropisetron increased SIRT1 gene expression and decreased phosphorylated FOXO3a/total FOXO3a protein ratios, NF-κB, and claudin-1 protein levels in the kidney of diabetic animals.

SIRT1 is a class III protein deacetylase which gets involved in the survival of the cells under stressful conditions by activating several key proteins. It is an essential factor for modulating gene expression in response to ROS generation such as NF-κB and foxo transcription factors (Brunet et al. 2004; Sengupta et al. 2011). Among foxo family, FOXO3a is assumed to have an important role during oxidative stress by upregulating several antioxidants, including SOD and CAT (Hasegawa et al. 2008; Kops et al. 2002). In particular, inactivation of FOXO3a has been associated with downregulation of CAT-induced oxidative stress during hyperglycemia condition (Wang et al. 2017b). SIRT1 manages the activation of FOXO3a by deacetylation and modulates the cell response to oxidative stress directly or indirectly (Hori et al. 2013).

It was declared that there is a functional association between SIRT1 and FOXO3 regulation of the MnSOD-mediated antioxidant defense system (Tanaka et al. 2009; Zhang et al. 2015). Accordingly, Zhang et al. (2015) demonstrated that icariin protected acute lung injury caused by intestinal ischemia/reperfusion-mediated SIRT1/FOXO3 signaling pathway by upregulation of the MnSOD expression.

Previous reports have also implied that upregulation of SIRT1 alleviates cisplatin-induced nephropathy, renal ischemic/reperfusion injury, and diabetic nephropathy (Funk and Schnellmann 2013; Gu et al. 2016; Kim et al. 2011). Notably, diabetes-induced downregulation of SIRT1 that drives the pathophysiological alterations leads to renal toxicity (Wang et al. 2017b). Hasegawa et al. (2013) demonstrated that decreased SIRT1 in proximal tubules triggered the initiation of DN and resulted in albuminuria. In line, the results of this research identified decreased renal SIRT1 gene expression in diabetic group. Therefore, activation of SIRT1 caused the resistance of renal tubular cells to cellular stress (Kume et al. 2013). All in all, SIRT1 activators have gained remarkable attention as interesting candidates for diabetes therapy (Song et al. 2018).

Moreover, the present research also showed marked inflammatory response in diabetic kidneys as evidenced by NF-κB elevation. It is an essential regulating factor linked to immune response and inflammation (Hayden and Ghosh 2012). Consistent with the findings of this research, several studies have reported that activation of NF-κB and its nuclear translocation increases in both experimental and human DN (Alzahrani et al. 2020; Kuhad and Chopra 2009). In addition, SIRT1 elevation contributes to NF-κB inactivation to rescue podocytes from injury in the diabetic kidney (Liu et al. 2014). Interestingly, SIRT1 overexpression attenuates a protein expression in tight epithelial junctions, namely, claudin-1, in hyperglycemic rats, which is in correlation with proteinuria level. Activated claudin-1 in podocytes deteriorates the glomerular barrier function via reducing synaptopodin or podocin expression resulting in albuminuria (Hasegawa et al. 2013). These findings were also approved in the kidneys of the diabetic rats.

Here, it was demonstrated for the first time that under diabetic conditions, tropisetron administration led to enhanced SIRT1 gene expression and concomitantly decreased phosphorylated FOXO3a/total FOXO3a protein ratios, NF-κB, and claudin-1 protein levels. Hence, it is very logical that such activation in SIRT1 and the subsequent decrease in the phosphorylated FOXO3a/total FOXO3a protein ratios, NF-κB, and claudin-1 to be, in part, may be responsible for the improvement in the diabetic kidney function.

Researchers reported that protective effects of tropisetron in DN appear to be 5-HT3 receptor independent since granisetron, another selective 5-HT3 receptor blocker, failed to protect DN. It was declared that antioxidative and anti-inflammatory mechanisms of tropisetron may have a critical role in renal tissue protection in diabetes (Barzegar-Fallah et al. 2015). Recently, our laboratory observed that tropisetron treatment increased insulin secretion and decreased blood glucose level by elevating GLUT2 gene expression as well as UCP2/ZnT8 pathway (Naderi et al. 2020). Tropisetron as a blocker of 5-HT3 receptor increased insulin release from pancreatic insulin-producing beta cell line which resulted in hypoglycemia (Heimes et al. 2009). Therefore, it seems that modulating insulin release and subsequently glucose homeostasis may involve in antioxidative and anti-inflammatory response contributing to kidney protection.

In line with the present findings, tropisetron reversed the oxidative damage by increasing SIRT1 gene expression in senescence in mice (Mirshafa et al. 2020). In addition, attenuation of liver injury, TNF-α, and IL-6 protein levels were observed in the liver of diabetic rats following tropisetron co-treatment (Amini et al. 2020; Gholizadeh-Ghaleh Aziz et al. 2019).

Also, in the present research, tropisetron showed a similar response to a standard drug, glibenclamide. Accordingly, glibenclamide exposure increased SIRT1 and decreased phosphorylated FOXO3a/total FOXO3a protein ratios, NF-κB, and claudin-1 levels in diabetic kidney in the current study. Glibenclamide is an established drug which has widely been used in the treatment of diabetic patients by inhibiting ATP-sensitive K+ channels. Since there have been some contradictory results regarding the glibenclamide effect in DN (Akbar et al. 2013; Elmalí et al. 2004; Nakamura et al. 2000), probably due to the dosage or treatment duration, this event provided the motivation for introducing a novel drug for diabetic patients as a safe and effective agent with fewer side effects.

Overall, tropisetron effectively improves renal function in DN probably by altering the expression of SIRT1, phosphorylated FOXO3a/total FOXO3a protein ratios, NF-κB, and claudin-1. This study had some limitations in supporting this subject. For example, morphological studies should attempt to provide further evidence to validate the present investigation. Also, the researchers did not employ molecule inhibitors to validate the exact signaling pathway mechanism.

Conclusion

In conclusion, the findings of this study showed the nephroprotective effect of tropisetron in STZ-induced T1DM. The noteworthy findings of this investigation indicated that tropisetron’s nephroprotective effect possibly involved changes in the expression of SIRT1, FOXO3a, NF-κB, and claudin-1. These data could be stimulating for further research in this area.

Author contributions

Mahrokh Samadi: data curation, formal analysis, and writing (original draft). Shiva Gholizadeh -Ghaleh Aziz: data curation. Roya Naderi: conceptualization, data curation, formal analysis, methodology, project administration, supervision, validation, visualization, writing (original draft), writing (review), and editing.

Funding

This study was supported by the Nephrology and Kidney Transplant Research Center, Urmia University of Medical Sciences, Urmia, Iran.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.