Protective Effects of Naringenin in a Rat Model of Sepsis-Triggered Acute Kidney Injury via Activation of Antioxidant Enzymes and Reduction in Urinary Angiotensinogen.

BACKGROUND Sepsis is a devastating medical condition. In the USA, about 745 000 people are diagnosed with sepsis annually. Although many anti-inflammatory drugs have been used to manage sepsis, the treatment success rate is very low. This study was undertaken to examine the protective effects of naringenin on sepsis-induced kidney injury in rats. MATERIAL AND METHODS Sepsis was induced in Wistar albino rats by cecal ligation and puncture methods. Histological analysis was performed with hematoxylin and eosin (HE) staining. Reactive oxygen species (ROS) levels were determined by flow cytometery. TUNEL assay was used to demonstrate apoptosis. Sandwich ELISA method was used for the determination of urinary angiotensinogen, and protein expression was determined by Western blot analysis. RESULTS We found that naringenin decreased atrophy in the glomerulus and enabled maintenance of the capsule area and normal tubular cavity of the septic rats. Admistration of naringenin at the dosage of 10 and 20 mg/kg to sepsis rats caused significant reduction in the sepsis-induced apoptosis of kidney cells, accompanied by decrease in Bax and increase in Bcl-2 expression. Moreover, naringenin also decreased the ROS levels in septic rats and downregulated the expression of SOD, CAT, and APX. The effects of naringenin were also examined on the levels of urinary angiotensinogen in sepsis rats. We found that naringenin caused a significant decrease in urinary angiotensinogen levels of septic rats. CONCLUSIONS Naringenin appears to have potential in the treatment of sepsis.

Background

Flavonoids include a large group of plant phytochemicals with many important pharmacological properties [1]. They are ubiquitously present in plant kingdom. Because of their structural properties, they can interact with a number of cellular entities such as proteins, enzymes, and nucleic acids [2]. They are strong antioxidants and are considered safe for human consumption [3]. Naringenin is a commonly occurring flavonoid with diverse activities, including anti-inflammatory, antioxidant, antimicrobial, and anticancer effects [4,5]. In this study, we for the first evaluated the effects of naringenin on sepsis-induced kidney injury. Sepsis is a devastating medical condition resulting from intense systemic inflammatory response to an infection, loss of blood, trauma, or neoplasm [6]. Owing its clinical presentations, it is often challenging to manage [7]. Sepsis may result in multiple organ dysfunction, shock, or even death. In critically ill patients, sepsis is one of the leading causes of death in USA [8]. Approximately 750 000 people in the USA are annually diagnosed with sepsis and of these, around 210 000 die [9]. Although several anti-inflammatory drugs are used to manage sepsis, the success rate is very low. It is believed that deciphering the molecular mechanisms underlying this disease may enable more effective treatment of the disease [10]. Here, we report that naringenin exerts protective effects on sepsis-induced kidney injury in rats and that naringenin helped to restore normal kidney anatomy in septic rats. Kidney injury induced by sepsis has been reported to be accompanied by induction of apoptosis in tubular regions of the kidney [11]. A commonly found flavonoid, naringenin, prevented apoptosis in the kidneys of sepsis model rats by preventing generation of reactive oxygen species and activation of ROS scavenging enzymes. Urinary angiotensinogen is considered an important biomarker of kidney injury [12]. In this study, we found that naringenin decreased the urinary angiotensinogen levels of rats, indicating the protective effects of naringenin against sepsis-induced kidney injury in rats.

Material and Methods

Animals and treatment grouping

Twenty 16-week-old Wistar albino rats were procured from the animal house of Shengli Oilfield Central Hospital. The rats were maintained under standard laboratory conditions with a light/dark cycle of 12 h. Food and water were available ad libitum. The rats were randomly divided into 4 groups. Group I (control) consisted of normal rats administered normal saline. Group II consisted of rats in which sepsis was induced by cecal ligation and puncture methods as descried previously [13]. Group III and Group IV consisted of septic rats administered 10 and 20 mg/kg of naringenin, respectively. The Animal Ethics committee of Shengli Oilfield Central Hospital approved the study (approval number SOCH/IV/996, 2018). At the end of the study, the animals were sacrificed by ketimine/xylazine anesthesia. The kidneys were extracted and stored at −80°C for histological analysis.

Determination of urinary angiotensinogen

Urine samples were collected into centrifuge tubes using metabolic cages to collect urine samples in centrifuge tubes. The urine samples were then centrifuged and then stored at −20°C for further experimentation. The urinary angiotensinogen level was determined by the sandwich ELISA method developed by Kobori et al. [14]. The pure recombinant proteins of rodent angiotensinogen were used as the standards, and absorbance was read at 450 nm.

Hematoxylin and eosin (HE) staining

The kidney specimens were washed in ice-cold saline. The tissues were then subjected to fixation in 10% formalin solution for 24 h. After embedding the tissues in paraffin, they were cut into 5-mm sections, followed by HE staining. Finally, the tissues were analyzed by a pathologist under a light microscope.

Determination of apoptosis and ROS levels

TUNEL assay was employed by using an apoptosis detection kit (Promega). Alexa Fluor 594-conjugated goat anti-mouse IgG (1: 500; Invitrogen) was used for TUNEL immunofluorescence. The glass slides were then mounted with cover slips with Vectashield mounting medium with DAPI. The slides were then observed under a fluorescence microscope and images were taken. The cells isolated from the kidney tissues from all the treatment groups were washed twice in PBS and re-suspended in 500 μl of DCFH-DA (10 μM) at 37°C in a dark room for 30 min. The samples were then examined instantly using flow cytometry as described previously [15].

Western blot analysis

The kidney tissues were lysed in lysis buffer containing the protease inhibitor. Around 45 μg of proteins from each sample were subjected to 10% separation, and then were transferred to a polyvinylidene difluoride (PVDF) membrane. Next, fat-free milk was used to block the membrane at room temperature for 1 h. Thereafter, the membranes were treated with primary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX) at 4°C overnight. Subsequently, the membranes incubated with rabbit secondary antibody (Santa Cruz Biotechnology, Inc., Dallas, TX). Finally, the signal was detected using the Odyssey infrared imaging system. Actin was used as a control for normalization.

Statistical analysis

All experimental procedures were performed in 3 biological replicates. The values obtained are presented as means of these 3 replicates ±SD. # P<0.05, * P<0.01 and *** P<0.001 were considered statistically significant. The statistical analysis was performed by t test using GraphPad prism 7 software.

Results

Naringenin prevents the sepsis-induced kidney injury

Histological analysis was carried out to assess the effects of naringenin on the kidney injury induced by sepsis in the rats. The Group 1 (normal) rats showed normal kidney histology with no glomerulus or renal tubule pathology (Figure 1A). The kidneys of septic rats (groups II) showed glomerulus atrophy, diffused capsule area, and enlarged tubular cavity (Figure 1B). However, treating rats with 10 and 20 mg/kg of naringenin significantly prevented or reversed the lethal effects of sepsis on kidney histology (Figure 1C, 1D). Naringenin administration also markedly decreased the damage to the kidney epithelial tissue.

Figure 1

(A–D) Histological analysis of kidney tissues of different treatment groups by HE staining. Group I (control) consisted of normal rats administered normal saline, Group II consisted of septic rats, and Group III and Group IV consisted of septic rats administered 10 and 20 mg/kg of naringenin, respectively. The experiments were performed in triplicate.

Naringenin prevented sepsis-induced apoptosis in kidneys

The effects of naringenin were also examined on the sepsis-induced apoptosis in the kidneys of rats by TUNEL and DAPI staining assays. The normal rats (Group I) rats did not showed any apoptosis, but the septic rats (Group II) showed high kidney cell apoptosis rates. We found that naringenin treatment at 10 and 20 mg/kg (Group III and IV) reduced the apoptosis of kidney cells (Figure 2). Additionally, the expression of Bax was increased and Bcl-2 was decreased in the septic rats (Group II) in comparison to the control rats (Group I), and treatment with 10 and 20 mg/kg (Group III and IV) of naringenin caused a decrease in Bax and an increase in Bcl-2 expression (Figure 3).

Figure 2

TUNEL assay and DAPI staining showing naringenin prevents apoptosis in kidney tissues. Group I (control) consisted of normal rats administered normal saline, Group II consisted of septic rats, and Group III and Group IV consisted of septic rats administered 10 and 20 mg/kg of naringenin, respectively. The experiments were performed in triplicate.

Figure 3

Expression of apoptosis-related proteins in different treatment groups as assessed by Western blot analysis. Group I (control) consisted of normal rats administered normal saline, Group II consisted septic rats, and Group III and Group IV consisted of septic rats administered 10 and 20 mg/kg of naringenin, respectively. The experiments were performed in triplicate and results are expressed as mean ±SD.

Naringenin caused decreased ROS levels in kidney tissues

As ROS has been implicated in sepsis-induced kidney injury [15], flow cytometery was used to examine the ROS levels in all 4 rat groups. The results showed that septic rats (Group II) had markedly higher levels of ROS levels as compared to the normal rats (Group I) (Figure 4). However, treatment with 10 and 20 mg/kg naringenin caused a remarkable reduction in the ROS levels in kidney tissues of septic rats (Group III and IV) (Figure 4). Taken together, these results suggest that naringenin inhibits sepsis-induced oxidative stress in kidneys.

Figure 4

Expression of antioxidant enzymes in different treatment groups as assessed by Western blot analysis. Group I (control) consisted of normal rats administered normal saline, Group II consisted septic rats, and Group III and Group IV consisted of septic rats administered 10 and 20 mg/kg of naringenin, respectively. The experiments were performed in triplicate and results are expressed as mean ±SD (# P<0.05 for Group I vs. Group II, * P<0.01 for Group II vs. Group III, and ** P<0.05 for Group II vs. Group IV).

Naringenin caused decreases in antioxidant enzyme expression

Next, to determine if the reduced ROS levels are triggered by activation of the antioxidant enzyme system, we performed Western blot analysis assessed to assess the effects of naringenin on expression of antioxidant enzymes (SOD, CAT and APX). The results showed that administration of naringenin at the dosage of 10 and 20 mg/kg to septic rats caused a significant increase in SOD, CAT, and APX expression levels (Figure 5).

Figure 5

Flow cytometric analysis showing ROS levels in different treatment groups. Group I (control) consisted of normal rats administered normal saline, Group II consisted septic rats. Group III and Group IV consisted of septic rats administered 10 and 20 mg/kg of naringenin, respectively. The experiments were performed in triplicates and expressed as mean ±SD (# P<0.05 for Group I vs. Group II, * P<0.01 for Group II vs. Group III and ** P<0.05 for Group II vs. Group IV).

Naringenin decreased the urinary angiotensinogen levels

Urinary angiotensinogen (UA) is an important biomarker of kidney injury [12]. We found that the levels of UA levels were significantly elevated in septic rats (Group II) as compared to normal rats (Group I) (Figure 6). However, administration of 10 and 20 mg/kg naringenin decreased the UA levels in rats in Groups III and IV, suggesting that naringenin decreased the sepsis-induced kidney injury.

Figure 6

Urinary angiotensinogen in different treatment groups. Group I (control) consisted of normal rats administered normal saline, Group II consisted of septic rats. Group III and Group IV consisted of septic rats administered 10 and 20 mg/kg of naringenin, respectively. The experiments were performed in triplicate and results are expressed as mean ±SD (# P<0.05 for Group I vs. Group II, * P<0.01 for Group II vs. Group III, and ** P<0.05 for Group II vs. Group IV).

Discussion

Sepsis is a common cause of death in intensive care units [7]. Sepsis may be accompanied by devastating medical complications such as multiple organ dysfunction and, ultimately, death [8]. Sepsis has also been reported to cause kidney injuries, and one of the main culprits is the generation of ROS [16]. Because of sepsis-induced mortalities, there is need for development of novel drugs for the treatment of sepsis. Natural products have always been a remarkable source of drugs. Microbes and plants have provided mankind with a number of drugs that are in use even today [17]. Naringenin is a plant-derived molecule with potent pharmacological potential. Because of the presence of naringenin in edible plants, it is considered safe for human consumption [18]. In the present study, we used a rat model of sepsis to evaluate the protective effects of naringenin, a common plant flavonoid. Since sepsis is often associated with kidney injury, the effects of naringenin were first examined on the kidney histology of the different rat groups, showing that sepsis disturbed the histology of the kidneys as evident from glomerulus atrophy, diffused capsule area, and enlarged tubular cavity. Naringenin treatment restored the normal kidney anatomy, showing the protective effects of naringenin against kidney injury induced by sepsis. A previous study has also shown the protective effects of naringenin against kidney injury in rats [19]. The kidney injury triggered by sepsis is also associated with apoptosis induction [11], and in the present study we found that naringenin inhibited the apoptosis induced by sepsis. Moreover, studies have shown that the development of sepsis is accompanied with generation of ROS and development of oxidative stress, which damages the cellular membranes and other cellular macromolecules [20]. Naringenin is an important antioxidant [21]. We found that naringenin caused a substantial decrease in the ROS levels as depicted by flow cytometery. Western blot analysis also showed a significant increase in the expression of the antioxidant enzymes. These results clearly show that naringenin reduces ROS levels and activates the enzymic antioxidant system of kidney cells. Studies have shown that urinary angiotensinogen is a vital indicator of kidney injury [12]. The present results show that naringenin decreased the levels of urinary angiotensinogen, indicating the protective effects of naringenin against sepsis-induced kidney injury.

Conclusions

We found that naringenin exerts protective effects against sepsis-induced kidney injury in a rat model by preventing apoptosis, reducing ROS levels, activation of the enzymic antioxidant system, and reduction of urinary angiotensinogen. Hence, naringenin may prove beneficial in the treatment of sepsis and deserves further evaluation.