Boswellic Acid and Carnosine Ameliorate Vanadyl-Sulfate-Induced Renal Damage via Regulating Nrf2/HO-1 and PI3K/AKT Pathways

TL;DR

Boswellic acid and carnosine help protect rat kidneys from damage caused by vanadyl sulfate by balancing key biological pathways.

Contribution

The study reveals that boswellic acid and carnosine reduce nephrotoxicity via Nrf2/HO-1 and PI3K/AKT pathways.

Findings

Vanadyl sulfate increased kidney damage markers like creatinine and blood urea in rats.

Boswellic and carnosic acids restored kidney function by reducing oxidative stress and fibrosis.

The protective effects were linked to modulation of Nrf2/HO-1 and PI3K/AKT pathways.

Abstract

Nephrotoxicity is a condition caused by the negative effects of several chemotherapy treatments on the kidneys. Our target was to determine how boswellic and carnosic acids protected rats against vanadyl sulfate (VOS)-induced nephrotoxicity. A total of 30 male Wistar albino rats were used in the investigation. They were divided into sex groups (five rats in each group) as follows: Rats in the control group were given carboxymethyl cellulose (CMC) at a concentration of 0.5%. VOS group was administered a weekly intraperitoneal injection of VOS 50 mg/kg for six consecutive weeks. For 6 weeks in a row, rats in the boswellic acid group were given injections of BA every day at a dose of 100 mg/kg orally. Group receiving carnosic acid received 100 mg/kg of CA orally daily for 6 consecutive weeks. For 6 weeks, rats in the boswellic acid plus VOS group were given 50 mg/kg of VOS intraperitoneally (i.p.) once a week and 100 mg/kg of BA orally daily. For 6 weeks, the carnosic acid plus VOS group received 50 mg/kg of VOS intraperitoneally once a week and 100 mg/kg of CA orally daily. In contrast to the control group, our results demonstrated that the injection of vanadyl sulfate to the VOS group contributed a significant increase in creatinine and blood urea as well as disordered kidney oxidative antioxidant interaction. The injection of vanadyl sulfate to the VOS group produced an increase in renal MDA concentration and a decrease in renal SOD activity, GSH content, GR, and CAT activities in comparison to the control group. When compared to the control group, our study on renal mRNA expression revealed that administering vanadyl sulfate to the VOS group increases renal iNOS, PI3K, and AKT mRNA expressions and modulates the mRNA expression of renal Nrf2 and HO-1. According to the current investigation, treating rats with BA or CA can reduce their nephrotoxicity from VOS; when the defective kidney oxidative antioxidant redox levels were returned to normal, the fibrosis in the Van-treated animals decreased. These findings are consistent with interactions between the Pi3k/Akt and Nrf2/Ho-1 pathways, as one of the human body’s most vital organs.

The online version contains supplementary material available at 10.1007/s12011-025-04786-9.

Figures

The chemical structure of carnosic acid [[14](#CR14)]

The chemical structures of *β*-BA and *β*-KBA [[12](#CR12)]

The experimental design

Changes of renal oxidative/ antioxidant redox in different groups. The impact on A MDA, B SOD, C GSH, D GR, and E CAT of vanadyl sulfate, boswellic acid, and carnosine in combination. The data (*n* = 5) were displayed as mean ± SE. With ANOVA and Tukey’s test as a post-ANOVA test, a and b, respectively, show a significant shift from the control and VOS groups at *p* < 0.05

The changes of renal mRNA expression of iNOS, Nrf2, HO-1, PI3K, and AKT in different groups. iNOS mRNA expression (A), Nrf2 mRNA expression (B), HO-1 mRNA expression (C), PI3K mRNA expression (D), and AKT mRNA expression (E) expression levels as influenced by Van, Bos, and Car and their combination. The data (*n* = 5) were displayed as mean ± SE. ANOVA with Tukey’s test as a post-ANOVA test shows that a and b represent significant differences from the control and VOS groups, respectively, at *p* < 0.05

The changes of renal protein concentration of iNOS, Nrf2, HO-1, PI3K, and AKT in different groups. iNOS protein concentration (A), Nrf2 protein concentration (B), HO-1 protein concentration (C), PI3K protein concentration (D), and AKT protein concentration (E) expression levels after Van, Bos, and Car have been combined. The bands on each group’s western blot (F). The mean ± SE was used to present the data (*n* = 5). ANOVA with Tukey’s test as a post-ANOVA test shows that a and b represent significant differences from the control and VOS groups, respectively, at *p* < 0.05

Tables

Table 2. The effect of vanadyl sulfate on serum urea and creatinine

	Urea (mg/dl)	Creatinine (mg/dl)
Control	20.48 ± 0.67	0.76 ± 0.04
Van	82.86 ± 2.1 a	5.8 ± 0.35 a
Bos	20.34 ± 1.28 b	0.79 ± 0.05 b
Car	21.62 ± 1.07 b	0.86 ± 0.02 b
Van + Bos	41.79 ± 1.25 a,b	2.05 ± 0.04 a,b
Van + Car	42.26 ± 1.06 a,b	1.93 ± 0.09 a,b

Full text

Introduction

The kidney, the most essential organ in the human body, aids in many essential functions, including detoxification, the elimination of harmful substances, homeostasis maintenance, and the regulation of extracellular fluid [1].

Acute kidney injury, or AKI, is a very common diagnosis in both hospital and pre-hospital settings, possibly impacting 60% of patients who are referred to an intensive treatment unit. Acute kidney injury has been worse by recent decades as a result of the growing use of drugs that can damage the kidneys, is the third most commonly caused by nephrotoxicity, according to epidemiological research. Up to 20% of severely unwell persons have been reported to employ nephrotoxic medicines [2].

The term nephrotoxicity refers to the rapid decline in kidney function caused by the negative effects of medications and substances. A number of different kinds exist, and certain drugs, like vanadium, may affect renal function in different ways [1].

When it relates to animals, vanadium (VOS) has a wide range of impacts. The ability of this metal to form organometallic compounds has led to a rise in research on the many chemical complexes and their diverse biological roles. Vanadium is found in low oxidation states in its organometallic compounds, but its most significant medical use is in the IV and V oxidation states as coordination compounds or polyoxovanadates [3]. Grain, parsley, dill seeds, black pepper, seafood, and mushrooms are all naturally high in vanadium [4]. It has been utilized as a micro-supplement for several human biological systems and is considered the third transition metal [5]. According to records, vanadium is one of the most crucial substances for blood glucose regulation and may even have anticancer properties [6] Furthermore, because vanadium compounds act as insulin mimetics, they have been acknowledged as physiologically relevant since the 1970s. Vanadium may be utilized as a therapy for diabetes mellitus, and its hypoglycemic activity has been studied in its physiological effects [7]. Furthermore, vanadium treatment demonstrated a strong impact on regulating a variety of metabolic disorders, with a particularly strong correlation shown with glucose homeostasis [8].

Among the chemicals produced from vanadium is vanadyl sulfate. It is noted to be a hypoglycemic drug because of its several positive, strong effects, including the provision of insulin by shielding the beta cells (β-cells) of the islets of Langerhans and stimulating the production of insulin by the β-cells [5].

Conversely, vanadium has a protracted withdrawal period that can last for many months following therapy and has a cumulative effect on several bodily organs and tissues. Vanadium’s harmful effects are exacerbated by this cumulative impact, particularly in the liver and kidneys. It alters the kidneys’ structure and function and increases hepatotoxicity by increasing reactive oxygen species (ROS) [9].

Several previous studies explained the renal damage induced by vanadium. Most hypotheses attributed the nephrotoxic effects of vanadium to the generation of ROS, which degrades DNA, denatures proteins, and disintegrates cell membranes [10]. This sets off a cascade of pro-inflammatory and vasoactive chemicals. These drugs made renal damage and fibrosis worse [11].

Around the world, natural products have attracted a lot of interest as potential medication candidates, and their application in drug research has grown to support conventional healthcare systems. Nearly 80% of the world’s population uses plant-based traditional health remedies because they are affordable and safe for use [12].

A lot of studies have been done on the use of natural treatments to avoid the negative effects of pharmaceuticals and drugs. Carnosic acid is one of the bioactive components of Rosmarinus officinalis (Fig. 1). Among its numerous biological functions are antioxidant, anti-inflammatory, antibacterial, anti-cancer, and anti-osteoclastic qualities [13].Fig. 1. The chemical structure of carnosic acid [14]

Carnosic acid, a phenolic diterpene, belongs to the family of secondary plant metabolites known as terpenoids, isoprenoids, or terpenes. When oxygen is available after the plant is collected and the leaves are dried, carnosic acid develops an oxidative derivative called carnosol (picrosalvin), an ortho-biphenolic diterpene. Lactone-structured phenolic diterpenes, such as 7-methyl-epirosmanol, rosmanol, and epirosmanol, are also produced by air exposure during the extraction process [15]. It demonstrated potent protective properties, including nephroprotective impact [16] and anticancer activity [17].

The main active components of the plant Boswellia serrata are boswellic acids or BAs. It has been used to treat a wide range of inflammatory conditions, both acute and chronic. These acids have proven to have potent anti-inflammatory effects in both in vitro and in vivo animal experiments. Their anti-arthritic, anti-rheumatic, analgesic, anti-diarrheal, anti-hyperlipidemic, anti-asthmatic, anti-cancer, antibacterial, hepatoprotective, and immunomodulatory qualities have also been reported [18].

For thousands of years, people from different cultures have used frankincense (pharmacological name: Olibanum Indicum), the common name for the gum resin of the Boswellia species, to cure a range of illnesses, including cancer and inflammatory diseases. Over 216 components have been identified in this resin thus far. Boswellic acids belong to the family of chemicals known as pentacyclic triterpenes (Fig. 2). The anti-inflammatory qualities of 11-keto-ß-boswellic acid (KBA) and O-acetyl-11-keto-ß-boswellic acid (AKBA) have been the main focus of recent studies [19].Fig. 2. The chemical structures of β-BA and β-KBA [12]

Although BAs and their derivatives have been shown to exhibit a wide range of bioactivities, such as anti-inflammatory, antioxidant, and anti-carcinogenic qualities, it is yet unknown what molecular mechanisms may be responsible for these benefits, being among the most potent anti-inflammatory components [20].

Numerous mechanisms and indications are used in the fight against AKI and renal oxidative damage. Numerous studies have demonstrated that nuclear factor erythroid 2-related factor 2 (Nrf-2) is a crucial cytoprotective mechanism that shields many cell types from oxidative stress. Nrf-2 controls the transcription of genes involved in cytoprotection, such as those encoding the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase, glutathione S-transferase, and catalase (CAT). Furthermore, there is evidence that Nrf-2 may function as a cytoprotective agent in animal models of several diseases, including renal IR damage and nephrotoxicity [21].

The intracellular phosphatidylinositol kinase phosphoinositide 3-kinase (PI3K) also exhibits serine/threonine (Ser/Thr) kinase activity. Akt is also known as protein kinase B or PKB. Tyrosine kinase first activates PI3K, which then transforms phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-triphosphate (PIP3). The accumulation of Akt at the plasma membrane is promoted by this mechanism. Next, 3-phosphoinositide-dependent protein kinase 1 (PDK1) aids in the phosphorylation of Akt’s Thr308. Activated Akt may carry out its biological activity in a variety of physiological processes, including cellular proliferation, differentiation, death, migration, and metabolism [22, 23].

As a downstream gene, induced nitric oxide synthase (iNOS) was discovered and modified by the MAPK signaling pathway. The majority of pro-inflammatory factors are regulated by NF-κB. For example, NF-κB can boost NO synthesis by triggering the downstream processes COX-2 and iNOS. The MAPK/iNOS signaling pathway is also involved in the control of necrosis. It was shown that IFN-γ activation of L929 cancer cells triggered the p38 MAPK signaling pathway, increasing the amount of NO content and iNOS expression and finally leading to cell necrosis. Moreover, it was observed that hydrogen sulfide may increase iNOS synthesis and trigger MAPK signaling pathways [24].

Although many previous studies discussed the nephrotoxic impact of VOS, the actual mechanism of nephrotoxicity is still unclear, and exploring protective mechanistic pathways against VOS-induced renal damage is still an urgent challenge. By quantitatively estimating the renal markers involved in kidney functions and assessing the impact of carnosic acid and boswellic acid on specific renal antioxidant and apoptotic key-regulating genes in the tissue of the kidney, we intended to evaluate these medicines’ ability to prevent Van-induced kidney injury.

Materials and Methods

Drugs and Chemicals

The materials utilized in this investigation were all acquired from Sigma-Aldrich Corporation. Vanadyl sulfate was purchased as a powder form (CAS No. 233706), boswellic acid (3-acetyl-11-keto-β-boswellic acid) as a powder form (A9855), and carnosic acid (C0609). The solutions of the used compounds were freshly prepared just before administration.

Animals

A total of 30 male Wistar Albino rats weighing 180–250 g were used. They are from an animal facility run by El Nahda University in Beni-Suef, Egypt. The rats were kept in metal cages with five rats each, inside a 12-h dark–light cycle, at room temperature (25 ± 2 °C), and with a relative humidity of 70%. Rats were fed on a standard diet (standard rodents ration) (Maka Co. for animal food and supplies, Beni Suef, Egypt) (proteins (18%), carbohydrates (40%), fats (4–10%), fiber (4–5%), vitamins, and minerals). Water is always provided ad libitum. The Faculty of Veterinary Medicine, Beni-Suef University, Egypt’s Experimental Animal Ethics Committee approved our current work, and all experimental procedures followed the National Institutes of Health’s (NIH) guidelines for the handling and care of lab animals. Approval (024–099).

Experimental Design

Oral administration of 10 mL/kg body weight of 0.5% CMC was administered to a rat in the control group by gavage. In VOS group, for 6 weeks in a row, rats received weekly injections of VOS; the dose was 50 mg/kg body weight intraperitoneally [9]. In the group of boswellic acid, rats were given 100 mg/kg of body weight of BA once a day for 6 weeks [25]. In carnosic group, for 6 weeks, rats were given 100 mg/kg of body weight of CA orally daily [26]. Rats in the boswellic acid + VOS group were given daily doses of BA (100 mg/kg orally) and weekly doses of VOS (50 mg/kg i.p.) for 6 weeks. Rats in the carnosic acid + VOS group were given 100 mg/kg of CA orally every day and 50 mg/kg of VOS intraperitoneally once every week for 6 weeks.

Blood samples were drawn from the eye’s medial canthus blood capillaries 24 h after the last injection and deposited in a 5-mL dried centrifuge tube. These tubes were incubated for 10 min at 37 °C after being left to coagulate for 5 min at an oblique angle. Observing the instructions presented to the testing groups for each measurement parameter, the neat sera were separated after 20 min of centrifugation at 1000 × g and stored in a deep freezer at − 80 °C until they were needed. After an overnight fast, the rats were sacrificed by being beheaded. The kidney tissues were separated into two sections after being gathered, weighed, and cleaned with phosphate-buffered saline (PBS). To make a uniform suspension, the first component was utilized. We suspended 0.5 g of kidney tissue in 5 mL of PBS (pH 7) and homogenized it using a homogenizer (Ortoalresa, Spain). To evaluate the oxidative and antioxidant properties of the kidneys, the supernatant was kept at − 80 °C. Following the guidelines provided by the real-time polymerase chain reactions (RT-PCR) and western blot analysis instruction kits, the second section was set aside for molecular studies (the western blot analysis and RT-PCR research) (Fig. 3).Fig. 3. The experimental design

Methods

Estimation of Serum Creatinine and Blood Urea

A colorimetric method, as described by Patton and Crouch [27], was employed to detect blood urea using the Sigma-Aldrich Urea Assay kits (Product no. MAK006, Sigma-Aldrich, Louis, MO, USA). The Sigma-Aldrich Creatinine Detection kit (Product no. MAK475, Sigma-Aldrich Chemical, Louis, MO, USA) was used to detect serum creatinine in accordance with Weissman et al. [28]. It can be either a colorimetric or fluorometric test.

Biochemical Investigations

Sigma-Aldrich Chemicals was located in St. Louis, MO, USA. According to Beutler’s method, the amount of GSH was measured using the GSH Assays kit (Product no. CS0260) [29]. The GR Test kit (Product no. GRSA) was used to assess GR activity in compliance with the Goldberg and Spooner [30] methodological approach. CAT activity was analyzed using the aid of the Aebi [31] approach and the CAT Assay Kit (Product no. MAK531). The Buege and Aust approach was employed to check the MDA concentration utilizing the MDA Test kit (Product no. MAK568) [32]. SOD activity was assessed with the SOD Activity Assay kit (Product no. MAK528) in compliance with the Nishikimi [33].

Western Blot Investigation for iNOS, PI3K, AKT, HO-1, and Nrf2 Measurements

The blotting method and the relevant antibody were used to measure the quantities of renal iNOS, PI3K, AKT, HO-1, and Nrf2. Proteins were first sorted based on their molecular weights using gel electrophoresis. This time, the TGX Stain-Free™ Fast-Cast™ Acrylamide Kit (Bio-Rad Laboratories, TNC, USA, Catalogue No. 161–0181) for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was used. The second stage was to create the stain-free cast (SDS-PAGE TGX) according to the manufacturer’s instructions. Clarity™ Western ECL substrate (Bio-Rad, USA cat#170–5060) was used to visualize the bands. To compare the bands’ intensity to that of β-actin, the ChemiDoc MP imager (Markham, Ontario, Canada, L3R8T4 Canada) was utilized, a software for image analysis [34, 35].

Determination of Real-Time Polymerase Chain Reaction (RT-PCR) Gene Expression for iNOS, PI3K, AKT, HO-1, and Nrf2

Kidney tissue was purified for RNA using the RNeasy Purification Reagent (Qiagen, Valencia, CA). AKT, HO-1, Nrf2, PI3K, and iNOS primers are presented in Table Table 2LE: Please check if table captions, entries, and note of Tables 1 and 2 are presented/edited correctly; otherwise, kindly amend. The effect of vanadyl sulfate on serum urea and creatinineUrea (mg/dl)Creatinine (mg/dl)Control20.48 ± 0.670.76 ± 0.04Van82.86 ± 2.1a5.8 ± 0.35aBos20.34 ± 1.28b0.79 ± 0.05bCar21.62 ± 1.07b0.86 ± 0.02bVan + Bos41.79 ± 1.25a,b2.05 ± 0.04a,bVan + Car42.26 ± 1.06a,b1.93 ± 0.09a,b1. Using real-time quantitative PCR, gene expression was evaluated in compliance with the SYBR Green I (Step OneTM, USA) criteria and Applied Biosystems version 3.1 software. Every detail is presented in connection with the β-actin gene [36]. Table 1. Primers for quantitative real-time PCRPrimer sequenceHO-1forward: 5′-TGCTAGCCTGGTGCAAGATA-3′reverse: 5′-GCCAACAGGAAGCTGAGAGT-3′Nrf2forward: 5′-CCTCAACTATAGCGATGCTGAATCT-3′reverse: 5′-AGGAGTTGGGCATGAGTGAGTAG-3′PI3Kforward: 5′-AACACAGAAGACCAATACTC-3′reverse: 5′-TTCGCCATCTACCACTAC-3′iNOSforward: 5′-CACCACCCTCCTTGTTCAAC-3′reverse: 5′-CAATCCACAACTCGCTCCAA-3′AKTforward: 5′-GTGGCAAGATGTGTATGAG-3′reverse: 5′-CTGGCTGAGTAGGAGAAC-3′β-actinforward: 5′-CCCATCTATGAGGGTTACGC-3′reverse: 5′-TTTAATGTCACGCACGATTT C-3′

Statistical Analysis

All collected data were illustrated as means ± standard error (SE). The results were evaluated by using SPSS 20 (SPSS, Chicago, IL, USA). One-way ANOVA followed by Tukey’s post hoc test was done to evaluate and compare the significance between testing groups. Values of p < 0.05 will be referred to as significant.

Results

The Effect of Vanadyl Sulfate on Kidney Functions

Our results in Table 2 showed that when compared to the control group, the VOS group’s levels of creatinine and serum urea significantly increased after receiving vanadyl sulfate (p = 0.0001). When Bos and Car were administered to both groups, serum urea and creatinine significantly decreased in the Van + Bos and Van + Car groups, respectively, compared to the VOS group (P = 0.0001). Furthermore, there was no significant change between the Bos and Car groups in contrast to the control samples (P = 0.0001) after administering boswellic acid and carnosic acid, respectively.

Means ± SE is used to express values. When compared to the control group, the superscript (a) letter indicates a significant difference, and when compared to the VOS group, the superscript (b) letter indicates a significant difference at p < 0.05.

The Effect of Vanadyl Sulfate on Renal Oxidative Antioxidant Redox

In Fig. 4, when vanadyl sulfate is administered to the VOS group, renal MDA concentration rises while renal SOD activity, GSH content, GR, and CAT activities decrease concurrently (p < 0.05) in contrast to the group under control. Compared to the VOS group, the administration of Bos and Car in both groups, Van + Bos and Van + Car, improved the renal oxidative/antioxidant redox by significantly lowering the renal MDA concentration and concurrently increasing the SOD, GSH, GR, and CAT activities (p < 0.05). Furthermore, when comparing the Bos and Car groups to the control group, there was no discernible change (p < 0.05) after administering boswellic acid and carnosic acid, respectively.Fig. 4. Changes of renal oxidative/ antioxidant redox in different groups. The impact on A MDA, B SOD, C GSH, D GR, and E CAT of vanadyl sulfate, boswellic acid, and carnosine in combination. The data (n = 5) were displayed as mean ± SE. With ANOVA and Tukey’s test as a post-ANOVA test, a and b, respectively, show a significant shift from the control and VOS groups at p < 0.05

The Effect of Vanadyl Sulfate on Renal mRNA Expression of iNOS, Nrf2, HO-1, PI3K, and AKT

According to our findings in Fig. (5), contrasted with the control group, the administration of vanadyl sulfate in the VOS group leads in a decrease in renal Nrf2 and HO-1 mRNA expression and an increase in renal iNOS, PI3K, and AKT mRNA expressions at p < 0.05. When Bos and Car are administered to both groups, renal Nrf2 and HO-1 mRNA expression rises in Van + Bos and Van + Car, respectively, but renal iNOS, PI3K, and AKT mRNA expressions fall concurrently when compared to the VOS group (p < 0.05). Furthermore, there was no discernible change between the Bos and Car groups in contrast to the group under control (P < 0.05) after administering boswellic acid and carnosic acid, respectively.Fig. 5. The changes of renal mRNA expression of iNOS, Nrf2, HO-1, PI3K, and AKT in different groups. iNOS mRNA expression (A), Nrf2 mRNA expression (B), HO-1 mRNA expression (C), PI3K mRNA expression (D), and AKT mRNA expression (E) expression levels as influenced by Van, Bos, and Car and their combination. The data (n = 5) were displayed as mean ± SE. ANOVA with Tukey’s test as a post-ANOVA test shows that a and b represent significant differences from the control and VOS groups, respectively, at p < 0.05

The Effect of Vanadyl Sulfate on Renal Protein Concentration of iNOS, Nrf2, HO-1, PI3K, and AKT

According to our findings in Fig. 6, contrasted with the control group, the administration of vanadyl sulfate in the VOS group resulted in a decrease in the concentration of renal Nrf2 and HO-1 proteins and an increase in the concentration of renal iNOS, PI3K, and AKT proteins at p < 0.05. Renal Nrf2 and HO-1 protein concentrations rise when Bos and Car are administered to both groups (Van + Bos and Van + Car, respectively), while renal iNOS, PI3K, and AKT protein concentrations fall concurrently when compared to the VOS group (p < 0.05). Furthermore, there was no noticeable distinction between the Bos and Car groups in contrast to the group under control (p < 0.05) after administering boswellic acid and carnosic acid, respectively.Fig. 6. The changes of renal protein concentration of iNOS, Nrf2, HO-1, PI3K, and AKT in different groups. iNOS protein concentration (A), Nrf2 protein concentration (B), HO-1 protein concentration (C), PI3K protein concentration (D), and AKT protein concentration (E) expression levels after Van, Bos, and Car have been combined. The bands on each group’s western blot (F). The mean ± SE was used to present the data (n = 5). ANOVA with Tukey’s test as a post-ANOVA test shows that a and b represent significant differences from the control and VOS groups, respectively, at p < 0.05

Discussion

Vanadium is essential for controlling the metabolism of cells. Vanadium has long been thought to be a highly effective treatment for diabetes and abnormalities in lipid metabolism [37]. There are several ways in which vanadium improves insulin levels in diabetics. The range of vanadyl compounds and oxidation states supports the anti-diabetic activity of vanadium, according to the research. Vanadium suppressed gluconeogenesis more significantly in kidney tissue than in liver tissue, according to Kiertszan et al. [38].

Vanadium has been shown to have several negative consequences and the potential to cause irreparable tissue damage, despite its ability to counteract oxidative stress. Vanadium’s harmful consequences include infertility, gastrointestinal problems, neurotoxicity, lung and kidney damage, and carcinogenesis [39].

We designed our investigation and gave VOS as a nephrotoxic drug to healthy, non-diabetic rats in an effort to bridge the gap between Van’s beneficial and detrimental effects. This demonstrated that VOS, not diabetes or metabolism, is the source of the developing nephrotoxicity [11].

Nephrotoxicity caused by vanadium treatment was originally documented in many previous studies. The development of tubular necrosis and increased urea and creatinine levels were indicators of renal impairment caused by VOS [40].

The administration of VOS in the current investigation results in significant renal impairment, as seen by a large rise in blood urea and creatinine levels. These findings align with those published by Shukla et al. [37].

Both extremely reactive non-radical oxygen derivatives and oxygen-derived free radicals, such as hydroxyl radicals, nitric oxide, and superoxide, are referred to as reactive oxygen species (ROS). ROS have both positive and negative effects on the body. Oxidative stress is a dangerous state caused by an unequal distribution of free radical generation and removal [41]. Oxidative stress is defined as an imbalance between the generation of ROS and the biological system’s ability to combat ROS or repair oxidative damage [42].

We found that administering VOS considerably reduced the levels of SOD and CAT activity. Vanadyl salt administration was linked to comparable cases of decreased SOD and CAT activity, according to Adebiyi et al. [43] and Ifukibot et al. [44]. Furthermore, the reduction in GSH content that we observed is in line with the findings of Mahmoud et al. in male rats given ammonium metavanadate [45]. In line with Adebiyi et al. and Ifukibot et al., who noted a considerable increase in MDA formation in male mice treated with sodium metavanadate [43, 44], our results demonstrate that VOS treatment considerably increases MDA in the VOS group. VOS may produce the reactive hydroxyl radicals H₂O₂ since it is a transition metal. This hypothesis is consistent with Bhattacharya’s theory that the presence of transition metals such as Fe^2^⁺ might raise MDA by producing H₂O₂ [41]. The notion that increased oxidative stress causes vanadium toxicity. Numerous earlier data strongly confirm this, which is linked to a lower degree of antioxidant protection [46].

Cell responses to oxidative stress are significantly influenced by nuclear factor erythroid 2-like (Nrf2) [47]. Toxins and carcinogens can be eliminated before they cause damage because Nrf2 regulates the transcription of several antioxidant genes that preserve detoxification genes and cellular homeostasis [48]. Nrf2 is the primary regulator of HO-1 transcription during oxidative stress. According to our findings, kidney Nrf2 levels decreased when VOS was administered. The overproduction of ROS in renal tissue, which also inhibits HO-1 expression, may be the cause of the decline in Nrf2 [49].

Despite being expressed by all cell types, Nrf2 migrates into the nucleus in response to increasing cellular stress. There, it stimulates the translation of target genes that encode proteins involved in iron metabolism, xenobiotic efflux, DNA repair, redox regulation, and protein homeostasis. As a result, Nrf2 is believed to support a number of anti-inflammatory characteristics, including the inhibition of NF-κB activity and a reduction in inflammatory mediators like iNOS. A previous study examined the use of drugs that increase Nrf2 to reduce ROS and halt the progression of renal diseases [47, 50].

AKT is activated by lipid kinases known as phosphoinositide 3-kinases (PI3Ks), which in turn control a number of biological processes. Therefore, it is believed that the PI3K/AKT pathway is necessary for the survival, differentiation, proliferation, and death of mammalian cells. According to Li’s research, PI3K can phosphorylate Akt to stop apoptosis. One important mechanism hypothesized to be involved in the prevention of apoptosis is the stimulation of the PI3K/AKT pathway [17].

Cellular malfunction and mortality result from increasing oxidative stress caused by increased ROS production. In both healthy and diseased circumstances, ROS are important in the induction of apoptosis. It is believed that the phosphoinositol-3-kinase PI3K/Akt signaling pathway is one of the ways that cells survive. According to studies, it is crucial for halting apoptosis brought on by oxidative stress [51].

The PI3K/Akt pathway is essential for cellular defense against inflammatory stimuli. Numerous investigations have shown that Nrf2 separates from Keap1 via a range of signal transduction pathways, including PI3K/Akt and that this process enables further signal transduction that triggers the activation of antioxidant enzymes. Furthermore, a number of studies have shown that PI3K/Akt signaling, which is essential for cell survival, is downregulated in response to prolonged oxidative stress. Controlling the PI3K/Akt signaling pathways might therefore be a helpful method to prevent ROS-induced apoptosis [52]. By blocking the dephosphorylation of the tyrosine-phosphorylated beta subunit, VOS was shown to directly affect the PI3K/AKT pathway. Consequently, this stops the activation of the PI3K-Akt pathway, which includes PI3K and Akt protein kinase. The metabolism of fats and carbohydrates depends on this mechanism [53].

The activity and stability of Nrf2, a transcription factor that initiates the cell’s antioxidant defense systems, are significantly regulated by the PI3K/AKT signaling pathway. The release of Nrf2 from its inhibitory protein, Keap1, is facilitated by the activation of PI3K and AKT. This enables Nrf2 to concentrate in the nucleus and triggers the production of genes that are cytoprotective and antioxidant. It has been demonstrated that elevated AKT activity greatly increases Nrf2’s basal activity and amplifies its reaction to oxidative stressors. This implies that by enhancing the Nrf2-mediated stress response, PI3K/AKT helps to protect cells. Conversely, Nrf2 activity is decreased when the PI3K/AKT pathway is inhibited, either by lowering its stability or by blocking its efficient nuclear translocation. Nevertheless, other pathways, such as MAPK or PKC, that are not dependent on PI3K/AKT can still activate Nrf2. These results show that although there are other ways to activate Nrf2, the PI3K/AKT pathway greatly promotes and supports its optimal function, particularly in oxidative stress situations. Targeting cellular antioxidant systems in the treatment and prevention of illness requires an understanding of this interaction [54].

Vanadium compounds are known to be very hazardous to renal parenchyma, despite their many benefits. Several studies looked on ways to prevent kidney impairment caused by Vans. We looked at a strong defense mechanism against kidney damage brought on by Van. Our study’s results were in line with previous research, indicating that more research on vanadium could be necessary before it is authorized for use in human medicine [40].

The main source of boswellic acid (BA), an extremely bioactive compound, is frankincense. It directly affects apoptosis and has potent anti-inflammatory and anticancer effects. Additionally, their biological efficacy against hepatitis, inflammation, arthritis, asthma, chronic colitis, and ulcerative colitis is supported by a multitude of data [12]. Blood urea and creatinine levels significantly decreased when BA was administered compared to the VOS group.

Plasma malondialdehyde (MDA) is an invaluable resource for identifying oxidative stress in pathogenic diseases. One important antioxidant enzyme, serum total superoxide dismutase (SOD), keeps cellular oxygen levels within physiological range by converting superoxide anion radicals that the body creates into hydrogen peroxide. Thus, SOD activity is a proxy for the body’s ability to eliminate reactive oxygen species [12]. Our findings suggest that BA can simultaneously reduce oxidative stress (ROS). MDA levels were substantially greater in VOS rats than in normal rats at the end of the treatment period, whereas CAT, GSH, GR, and SOD activity were significantly lower. Oral BA therapy significantly increased blood levels of CAT, GSH, GR, and SOD while lowering serum MDA levels in comparison to the VOS group. Moreover, BA significantly reduced renal iNOS expression due to its ability to reduce nitrogen species formation, NO synthesis, and iNOS overexpression [25].

By enhancing the Nrf2/HO-1 pathway, which increases the expression of both Nrf2 and HO-1, BA offers protective benefits in a range of oxidative stress models [55]. BA may modify the Nrf2/HO-1 pathway through a number of underlying mechanisms. BA may increase Nrf2’s nuclear translocation, which in turn increases its transcriptional activity [56]. After this upregulation, a feedback loop that further strengthens cellular resistance to oxidative stress is facilitated by the increased HO-1 synthesis.

For example, in injured renal tissues, BA therapy has been demonstrated to decrease indicators of oxidative damage and restore antioxidant enzyme activity [57]. This implies that by strengthening the cellular antioxidant defense mechanism, BA may mitigate nephrotoxicity. In our investigation, we observed an increase in alterations in the levels of HO-1 and Nrf2 mRNA expression in the renal tissues after BA administration, suggesting that BA can influence these defense mechanisms. This is consistent with results from earlier research that show that BA protects against different types of tissue damage by upregulating Nrf2 and HO-1 [58].

The PI3K/AKT pathway regulates a number of biological processes, including cell development, survival, and death; dysregulation of this system is frequently linked to cellular damage and apoptosis [59]. BA has been shown to decrease the activity of PI3K/AKT, thereby inhibiting damage and apoptosis [60]. It has been established that ROS can activate the PI3K/AKT pathway, which in turn causes apoptosis and cell death [61].

Research has demonstrated, for instance, that BA can reduce AKT phosphorylation, a crucial step in the activation of this signaling pathway [62] and that downregulating the PI3K/AKT pathway protects against renal apoptosis [63].

Among the many biological properties of CA are its anti-inflammatory, anti-carcinogenic, and antioxidant properties. The compound’s potential as a therapeutic agent has been clarified by a study on its mechanism of action, which has been spurred by these features [64].

Compared to the Van group, our investigation showed that the nephroprotective effect of CA was exhibited by a reduction in creatinine and blood urea levels. In the treatment of rheumatoid arthritis, CA lowers inflammation and oxidative stress by inhibiting cellular lipid peroxidation [65], lowering MDA, and triggering the activity of cellular antioxidant enzymes, including SOD and GSH-Px [66]. CA inhibited the production of pro-inflammatory chemicals, such as iNOS [67].

According to reports, CA strengthens cells’ defense mechanisms against oxidative damage by activating the Nrf2/HO-1 signaling pathway. For example, research has demonstrated that CA increases HO-1 expression and facilitates Nrf2’s nuclear translocation in a variety of oxidatively stressed cell types [66]. According to a recent study, CA treatment dramatically raised HO-1 expression in oxidatively stressed renal cells, which decreased apoptotic and cellular damage indicators [68].

CA is also known to regulate the PI3K/AKT signaling pathway, which lowers inflammation and increases cell survival, in addition to its effect on the Nrf2/HO-1 pathway [69]. This implies that CA improves kidney regeneration by encouraging survival signals in renal cells in addition to providing protection against oxidative damage.

Since oxidative stress is known to stimulate the PI3K/AKT pathway, CA’s potent antioxidant properties may reduce it [70]. CA may reduce inflammation and cellular damage by indirectly inhibiting PI3K/AKT signaling through the scavenging of reactive oxygen species (ROS) [71]. It has been demonstrated that CA reduces endothelial cell AKT activation, which lowers inflammation and enhances endothelial function [72]. Furthermore, CA suppresses irregular cell proliferation, a defining feature of atherosclerosis and restenosis, by blocking PI3K/AKT signaling in the vascular system’s smooth muscle [71].

Conclusion

Overall, the current study demonstrates for the first time that BA and CA dramatically reduce the risk of vanadyl-sulfate-induced kidney damage by regulating the Nrf2/HO-1 and PI3K/AKT signaling pathways. Both substances protect the kidneys by activating the Nrf2 pathway. They promote renal cell survival and function by lowering oxidative stress levels and increasing the antioxidant response. Moreover, as the PI3K/AKT pathway is crucial for controlling cell survival, death, and proliferation, focusing on it is another factor contributing to its therapeutic potential. In a way, this suggests that BA and CA have the ability to be strong modulators of cellular stress responses, which might make them useful therapeutic agents for treating renal damage brought on by pharmacological or environmental insults like vanadyl sulfate. To maximize their use in renal protection and other oxidative stress-related disorders, more investigation into the specific molecular mechanisms, including possible synergistic effects and clinical implications, will be necessary.

Electronic Supplementary Material

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