Protective Effect of Betulinic Acid Administration on Kidney Protective Effect of Betulinic Acid Administration on Kidney Damage in Acetaminophen-Induced Nephrotoxicity Model Damage in Acetaminophen-Induced Nephrotoxicity Model

Background : Acetaminophen (APAP) is the most widely used analgesic drug worldwide, but it may induce renal toxicity. Betulinic acid (BA) ameliorates the oxidative stress and inflammatory response to renal damage. The present study aimed to investigate the potential protective effects of BA treatment through an experimental kidney damage rat model administered with APAP. Methods : Sprague – Dawley male rats were randomly divided into four groups: control, BA (25 mg/kg for 15 days), APAP (1 g/kg), and APAP + BA groups. BA was administered via oral gavage at a dose of 25 mg/kg for 15 days. APAP was dissolved in hot saline and administered on the last day to produce nephrotoxicity via a single oral gavage at a dose of 1 g/kg. Kidney tissue samples were analyzed for human cartilage glycoprotein 39 (YKL-40), kidney injury molecule 1 (KIM-1), interleukin 18 (IL-18), superoxide dismutase (SOD), and malondialdehyde (MDA). Data were subjected to one-way analysis of variance and the Wilcoxon rank-sum test Results : Renal tissue YKL-40, KIM-1, IL-18, and MDA levels in the APAP group were significantly higher than those in the control group ( p < 0.05). The BA treatment completely restored renal KIM-1, YKL-40, and MDA levels and partially restored renal IL-18 and SOD levels in the rats subjected to renal damage induction ( p < 0.05). The intertubular regions of rats administered with APAP had degeneration, necrosis, and infiltration of inflammatory cells and were immunopositive for IL-1 beta and 8-hydroxy-2′ - deoxyguanosine. Conclusions : BA can be used in the prevention and replacement treatment of nephrotoxicity due to its inhibitory properties in multiple pathways and powerful antioxidant effects.


I N T R O D U C T I O N
Acetaminophen (APAP) is one of the most widely used analgesic drug worldwide despite its capability to induce renal toxicity at overdose.Metabolites from APAPglutathione and APAP-cysteine conjugates have been implicated in renal toxicity. 1APAP transforms into the harmful p-aminophenol metabolite, which is a nephrotoxin in the kidney that initiates tissue damages, specifically tubular and cortical necrosis. 2In addition to APAPinduced oxidative stress and renal damages, 3 the activation of inflammatory and oxidative mediators contributes to nephrotoxicity. 4e principal response to kidney injury is the development of inflammation and oxidative stress, which can lead to renal fibrosis.Biomarkers associated with renal inflammation and repair may be helpful in distinguishing patients at risk of kidney damage from those who are likely to recover. 5 Biomarkers are frequently examined to diversify risk prediction, in addition to the usual clinical variables, and to explore potential pathological mechanisms that may be targets for future intervention.Human cartilage glycoprotein 39 (YKL-40), a member of "mammalian chitinase-like proteins," is secreted by macrophages, neutrophils, chondrocytes, endothelial cells, vascular smooth muscle cells, and cancer cells and also known as chitinase-3-like protein 1.It is a novel biomarker for acute and chronic inflammation.A high serum YKL-40 concentration indicates poor prognosis in a wide range of diseases and clinical conditions. 6,7e kidney can effectively recover from an ischemic or toxic insult that results in cell death as the tubular epithelium can proliferate and replace lost cells.The balance between injury and repair is the key determinant of the fate of the injured kidney.Potent factors induced by injury facilitate adaptive repair. 8Kidney injury molecule 1 (KIM-1), a transmembrane glycoprotein, is upregulated more than any other protein in the proximal tubule of the kidney with various forms of injury.KIM-1 is a phosphatidylserine receptor that mediates phagocytosis of apoptotic bodies and oxidized lipids.The chronic expression of KIM-1 results in progressive kidney fibrosis and chronic kidney failure, which are possibly related to its phagocytic function to take up noxious compounds, such as oxidized lipids. 9,10e role of inflammation in nephrotoxicity has been increasingly appreciated with the involvement of leukocytes, adhesion molecules, chemokines, and cytokines.Interleukins (ILs) are important mediators of the immune reaction in the innate immune system response and adaptive immunity.IL-18 is a proinflammatory cytokine involved in the pathogenesis of acute kidney damage. 11,12tulinic acid (BA) is mainly isolated from plant sources, such as birch, eucalyptus, and plane trees. 13It's potent physiological and pharmacological activities are related to anti-diabetic, antitumor, anti-inflammatory, and antibacterial effects.BA can help protect cells against oxidative damage by reducing the oxidative stress caused by free radicals.These antioxidant effects can potentially mitigate the negative effects associated with aging, inflammation, and certain diseases.BA exerts a strong anti-inflammatory effect by inhibiting inflammation.BA can also reduce the excessive response of the immune system and regulate the production of inflammatory cytokines. 14,15The present study was conducted to determine whether BA has protective effects on experimentally induced kidney damage using a rat model administered with APAP.

Animals and experimental design
Ethics committee approval of the study was obtained from Atatürk University Local Ethics Committee for Animal Experiments.Male Sprague-Dawley rats (N = 32, 200-250 g, and 6-8 weeks old) from the Experimental Animal Laboratory of Atatürk University were housed in special rooms with an ambient temperature of 22°C ± 2°C and humidity of 50%-60% under a photoperiod of 12:12 h light:dark conditions.The animals were given tap water and standard diet ad libitum.The rats were randomly divided into four groups (n = 8 per group): (1) control, (2) BA, (3) APAP, and (4) APAP + BA groups.BA (Sigma Chemical Co., St. Louis, MO) was administered via oral gavage at a dose of 25 mg/kg for 15 days. 16APAP (Sigma Chemical Co., St. Louis, MO) was dissolved in hot saline and administered on the last day to induce nephrotoxicity via a single oral gavage at a dose of 1 g/kg. 17APAP was administered 24 h after the last BA treatment.

Biochemical examination
Serum BUN, creatinine, ALT, and ALP were analyzed using an autoanalyzer (RX Monaco; Randox Laboratories Ltd., County Antrim, UK).The method of Sun et al. 18 was used to determine the SOD activity (U/mg protein) in kidney tissue samples; the method is based on the utilization of the xanthine oxidase system as a superoxide generator and inhibition of nitroblue tetrazolium reduction.Renal MDA levels (nmol/mg protein) were determined by the method of Esterbauer and Cheeseman 19 based on the measurement of the absorbance of the pink complex that formed in the presence of thiobarbituric acid at a wavelength of 532 nm.
Renal KIM-1, YKL-40, and IL-18 levels (pg/mg) were measured by enzyme-linked immunoassay (ELISA; ELK Biotechnology Co., Ltd, Wuhan East Lake Hi-Tech Development Zone, Hubei, China), in accordance with the manufacturer's instructions.In the ELISA method, an antigen immobilized on a solid surface was complexed with an antibody linked to an enzyme.Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product at 450 nm.

Histopathological examination
The kidney tissue samples were fixed in 10% buffered formalin and routinely processed for histological examination by embedding in paraffin wax.The tissue sections were cut at 4 µm thickness and stained by hematoxylin-eosin (HxE) for observation under a light microscope (Olympus Bx51 with a DP72 camera system, Olympus Corp., Tokyo, Japan). 20ch specimen was examined in 10 randomly selected areas of an X40 objective.The scores for hyperemia, degeneration, necrosis, and inflammatory cells were derived semi-quantitatively using light microscopy on the preparations from each rat and reported as follows: Grade 0 = -(negative); Grade 1 = +1 (mild); Grade 2 = +2 (moderate); Grade 3 = +3 (severe); Grade 4 = +4 (most severe). 21

Statistical analysis
The data were analyzed using a commercial software (Statistical Analysis of the System (SAS), Version 9.0, SAS Institute Inc., Cary, NC). Biochemical parameters (continuous data) were subjected to one-way analysis of variance using the GLM procedure.The group mean differences were elucidated by the least-significant difference option.Histopathological and immunohistochemical parameters (discrete data) were subjected to the Wilcoxon rank-sum test using the NPAR1WAY Procedure (SAS).Values at p ≤ 0.05 were considered significant.

Kidney injury markers
The APAP administration group caused 4.1-, 2.7-, 3.3-, and 2.5-fold increases in concentrations of BUN, creatinine, ALT, and ALP, respectively, compared with the control group (p < 0.05, Table 1).Except for that of ALP, the BA treatment reduced BUN, creatinine, and ALT concentrations in the APAP group to the levels of the control group.Compared with the control group, the APAP administration increased renal KIM-1, YKL-40, IL-18, and MDA levels by 117%, 127%, 228%, and 100%, respectively (p < 0.05, Table 2) and decreased renal the SOD level by 64% (p < 0.05, Table 2).The BA treatment completely restored renal KIM-1, YKL-40, and MDA levels and partially restored renal IL-18 and SOD levels in the rats subjected to renal damage induction.

Tissue analysis
Nephrotoxicity induction by APAP administration was successful, as reflected by the dramatic increases in the scores for hyperemia, degeneration, necrosis, and infiltration of inflammatory cells (macrophages and lymphocytes) in the intertubular region (Table 3; Figures 1A, 1B, and 1C).BA treatment was partially effective in ameliorating these histopathological parameters (Table 3; Figure 1D).

Biomarker expression
Responses to immunopositivity for IL-1β (Table 3; Figure 2) and 8-OHdG (Table 3; Figure 3) among the experimental groups were similar to the histopathological findings.APAP administration resulted in immunopositivity for IL-1β and 8-OHdG of the kidney tubular epithelial cells, mesengial cells, and endothelial cells of blood vessels, which were partially alleviated by the BA treatment (Table 3; Figures 2 and 3).

D I S C U S S I O N
APAP is one of the nonsteroidal anti-inflammatory drugs and has a widespread clinical use.When it is used at high doses, however, APAP can cause acute toxicity in organs involved in its metabolism and elimination, particularly in the liver and kidneys. 22Kidneys are dynamic organs having important roles in maintaining body homeostasis, water, and acid-base and electrolyte balance.Some biochemical and physiological properties of the kidney make it more susceptible to ischemic and toxic damage compared with other organs. 23This experiment questioned whether BA treatment alleviates APAPinduced nephrotoxicity using biochemical and histopathological markers pertinent to kidney damage.
Many studies indicated elevated BUN and creatinine levels as the evidence of renal dysfunction. 24Naguib et al. 25 and Das et al. 26 reported increases in BUN and creatinine levels upon the hepatorenal damage induction by paracetamol.In agreement with previous studies, APAPinduced nephrotoxicity was associated with elevations in BUN, creatinine, ALT, and ALP concentrations.APAP is a nephrotoxin and can induce nephrotoxicity at a single dose of its administration. 27,28In APAP intoxication, increased levels of N-acetyl-p-benzoquinoneimine lead to increased formation of superoxide radicals (O2 -) and hydrogen peroxide (H2O2). 29Kidney damage induced by nephrotoxins may result in chronic renal failure, nephrotic syndrome, tubular dysfunction, and acute renal failure due to triggering by oxidative stress. 23,30MDA, an important indicator of oxidative stress, is formed by the peroxidation of polyunsaturated fatty acids.Lipid peroxidation is the chemical reaction initiated by free radicals and involves the oxidation of polyunsaturated fatty acids on the membrane structure.Reactive Makara J Health Res.
April 2023 | Vol.27 | No. 1 aldehydes, including MDA, cause cross-linking and polymerization in membrane components, which eventually leads to alterations in membrane functions, involving deterioration in membrane structure, ion transport, and enzyme activity. 31SOD is an endogenous antioxidant enzyme protecting the cells responsible for oxygen metabolism from the adverse effects of superoxide radicals.In the literature, SOD activity was shown to decrease in acute kidney injuries. 32,33Double MDA concentration and decreased SOD concentration in the APAP group can be attributed to the lipid peroxidation reaction and decreased antioxidant protection system success of renal damage.BA has been suggested to exhibit antioxidant properties, thus attenuating the development of oxidative stress in experimental animal models.BA has antioxidant effects, which can protect kidney cells from oxidative damage caused by free radicals.Oxidative stress contributes to kidney damage.Thus, the antioxidant properties of BA may support kidney health.BA can help protect cells against oxidative damage by reducing MDA levels and inducing SOD activity.These effects can potentially mitigate the negative effects associated with kidney damage. 34,35vere inflammatory response also occurs in kidney damage resulting from oxidative stress. 36The study by Ucar et al. 37 , in which kidney damage was induced by APAP administration, revealed increases in the levels of inflammatory cytokines.Inflammation underlies the pathogenesis of many acute or chronic kidney diseases, and IL-18 is a useful marker for the diagnosis of diseases and prediction of their severity and prognosis. 38,In agreement with the study of Shen et al. 39 , a dramatic increase in IL-18 concentration was observed in rats exposed to kidney damage upon APAP administration.
KIM-1 is found at very low levels in the proximal tubules of the normal kidney.However, its level increases in damaged epithelium. 40,41It is one of the proteins most induced in the kidneys following acute kidney injury 42,43 and considered a prominent biomarker. 44The KIM-1 response upon APAP administration is similar to those reported by Kim et al. 45 and Coban et al. 28 We observed the positive effects of BA on KIM-1 in our study.No research investigated the relationship between BA and KIM-1.However, some studies suggested that BA may have a protective effect on kidneys by reducing oxidative stress 46 and inflammation 47 , which can lead to decreased KIM-1 levels.KIM-1 is a biomarker used to detect early signs of kidney injury, and its reduction may indicate improved kidney function.Further research is needed to fully understand the potential relationship between BA and KIM-1.
YKL-40, identified as a new inflammatory marker, plays an important role in inflammation, angiogenesis, extracellular matrix remodeling, and fibrosis development. 48,49uthumana et al. 50reported that YKL-40 levels increased to limit kidney damage and support adaptive repair mechanisms.The YKL-40 level became more than doubled in the APAP group, which was accompanied by increased cytokine levels because inflammatory cytokines stimulate the synthesis. 51,52YKL-40 is a marker of inflammation and tissue remodeling and associated with various pathological conditions, including kidney diseases.However, no current evidence suggests that BA has a direct effect on YKL-40 levels or activity.In our study, we observed the positive effects of BA on YKL-40.We believe that the positive effect of BA on YKL-40 levels resulted from its capability to halt the inflammatory process.Decreased inflammation may have led to a decrease in YKL-40 levels.Further research is needed to explore any potential relationship between BA and YKL-40 in kidney disease or other conditions.
The APAP-induced renal damage was evident, as reflected by histopathological findings, such as degeneration in kidney tissues and necrosis and hyperemia in epithelial cells.Moreover, immunohistochemical findings revealed the increased expressions of IL-1β and 8-OHdG.
Inflammation and oxidative stress play important roles in the pathophysiology of many kidney diseases and their complications. 53Acute kidney injury, as a result of longterm use of APAP, is one of the most common conditions.Natural ingredients with high antioxidant potency may help protect renal tissues from the APAP-induced nephrotoxicity.
BA may have various positive effects on kidneys.Some studies suggest that BA can help the kidneys function in a healthy way by protecting kidney cells from oxidative stress and inflammation. 46,54In addition, BA is being investigated as a potential treatment option for some kidney diseases, such as kidney inflammations and kidney stones.Khataylou 54 showed that BA markedly decreased the production of proinflammatory cytokines.
BA supplementation provides nephron-protective effects through ameliorating oxidative stress and inflammatory response. 55Lingaraju et al. 55 reported that BA administration increased renal antioxidant activity by suppressing inflammatory cytokines.BA reduced oxidative stress and inflammation in renal tissue.An experimental study examining the effects of BA on renal fibrosis reported that BA prevents fibrosis by inhibiting nuclear factor-κB activation. 56The BA treatment completely and partially ameliorated the serum parameters.These positive effects on metabolic profile were accompanied by an enhanced antioxidant status.The beneficial effects of BA treatment were also notable in histopathological and immunohistochemical evaluations.

C O N C L U S I O N S
Acute APAP exposure generated inflammation and oxidative stress in tissues, as reflected by the increases in April 2023 | Vol.27 | No. 1 specific (KIM-1, YKL-40, and IL-18) and general (SOD and MDA) biomarkers involved in the pathophysiology of nephrotoxicity.Overall, BA treatment was partially effective in ameliorating renal damage.Studies investigating the effect of BA on kidney injury are limited.Our study is one of the few research investigating the effect of BA on kidney injury, which makes the topic innovative and interesting.Our study can contribute new information to the literature regarding the effect of BA on kidney injury.This can provide an important scientific contribution for researchers and healthcare professionals.The study results suggested that BA ameliorated the oxidative stress and inflammatory response of APAP-induced renal damage by reducing the KIM-1/YKL-40/IL-18 signaling pathway.Future research should consider different treatment dosages and durations of BA treatment for kidney injury.

C O N F L I C T O F I N T E R E ST
The authors declare that they have no known competing financial interests nor personal relationships that could have appeared to influence the work reported in this paper.

F U N D I N G
least square means ± standard error (SE).Different superscripts within columns differ (p < 0.05). 1 BUN = blood urea nitrogen; ALT = alanine aminotransferase; ALP = alkaline phosphatase.2BAwas administered via oral gavage at a dose of 25 mg/kg for 15 days (Nader and Baraka 2012).The APAP, after its dissolution in hot saline, was administered on the last day to produce nephrotoxicity via a single oral gavage at a dose of 1 g/kg (Dokumacioglu et al. 2017).

FIGURE 1 .
FIGURE 1.Effect of BA treatment on the histopathology of renal tissue in rats exposed to APAP-induced nephrotoxicity.HxE, Bar: 50 µm.A-B) No histopathological lesions in the healthy rats not given BA (control) and given BA (BA group).C) Inflammatory cell infiltration, degeneration, and necrosis in the epithelial cells of proximal tubules in rats with APAP-induced nephrotoxicity (APAP group).The star on the figures indicates inflammatory cell infiltration, and the black arrow shows degeneration and necrosis.D) Moderate histopathological changes were observed in rats with APAP-induced nephrotoxicity and treated with BA (Group APAP + BA).

FIGURE 2 .
FIGURE 2. Effect of BA treatment on the immunohistochemistry of IL-1β expression in rats exposed to APAP-induced nephrotoxicity.Immunopositive stained epithelial cells, mesengial cells, and endothelial cells of blood vessels.The star on the figures indicates inflammatory cell infiltration, and the black arrow shows degeneration and necrosis.Bar: 50 µm.

FIGURE 3 .
FIGURE 3. Effect of BA treatment on the immunohistochemistry of 8-OHdG expressed in rats exposed to APAP-induced nephrotoxicity.Immunopositive stained epithelial cells, mesengial cells, and endothelial cells of blood vessels.The star on the figures indicates inflammatory cell infiltration, and the black arrow shows degeneration and necrosis.Bar: 50 µm.

TABLE 1 .
Effect of BA treatment on serum biochemistry profile of rats exposed to APAP-induced nephrotoxicity *

TABLE 2 .
Effect BA was administered via oral gavage at a dose of 25 mg/kg for 15 days (Nader and Baraka 2012).APAP, after its dissolution in hot saline, was administered on the last day to produce nephrotoxicity via a single oral gavage at a dose of 1 g/kg (Dokumacioglu et al. 2017).

TABLE 3 .
Effect of BA treatment on the histopathology and immunohistochemistry of renal tissues from rats exposed to APAPinduced nephrotoxicity * This work was supported by Coordinator of Scientific Research Projects [2021.M84.02.02] at Artvin Coruh University.Elgamal M, Abdelaziz A, Ellithy M, Magdy D, Ali L, et al.Toll-like receptor 4 blocker as potential therapy for acetaminophen-induced organ failure in mice.Exp Ther Med.2015;10(1):241-6.3.Ghosh J, Das J, Manna P, Sil PC.Acetaminophen induced renal injury via oxidative stress and TNF-α production: therapeutic potential of arjunolic acid.Toxicology.