An abrupt reduction in renal function is the hallmark of acute kidney injury (AKI), which occurs in 10-15% of hospitalizations and affects >50% patients in intensive care in the United States (1,2). Acute tubular necrosis, which is the most common cause of AKI, may result from ischemia, exogenous nephrotoxic agents (such as iodinated contrast media, aminoglycosides, amphotericin B and vancomycin) or endogenous nephrotoxic damage due to rhabdomyolysis and hemolysis (2). In particular, ischemia can arise in the kidney after various urological treatments, including kidney transplantation, partial nephrectomy and renal artery surgery (3,4). Additionally, trauma, shock and sepsis are also amongst the most commonly reported causes of kidney ischemia (3-5). Ischemia is considered to be one of the primary causes of AKI (6). Renal damage first occurs during ischemia, which is then exacerbated by the restoration of blood flow (3). Diagnosis of renal injury caused by ischemia/reperfusion (I/R) depends on clinical assessment, urinary or blood biochemical markers, radiological findings and eventually histologic examination (7,8). Blood urea nitrogen (BUN) and serum creatinine are useful key markers of renal I/R injury (8). CT is not applicable for renal I/R injury diagnosis due to the contrast toxicity seen in acute tubular necrosis (ATN) (7). Although MRI with contrasts can exert toxic effects on kidney functions, T₁ and T₂-weighted MRI can provide useful information regarding the extent of damage induced by hypoxia (7,8). Histopathologically, I/R injury frequently manifests as damage to the tubular epithelium, primarily due to the high energy demands of renal tubules. This can result in ATN and/or AKI (9).

Alterations in the mitochondrial oxidative phosphorylation system during ischemia results in decreased adenosine triphosphate (ATP) and antioxidant production (10). Furthermore, disruption of ion pumps (Na⁺/K⁺/ATPase, Na⁺/H⁺, Ca2⁺/ATPase pumps) leads to the accumulation of hydrogen, sodium and calcium ions, resulting in cell swelling and the activation of proteases (such as apoptosis protease-activating factor) and phosphatases (such as serine/threonine-protein phosphates) in the cytoplasm (4,10-12). Activated enzymes then degrade the cytoskeleton and membrane phospholipids, resulting in the production of reactive oxygen species (ROS). With the resupply of oxygen following reperfusion, ROS are produced by the xanthine oxidase system (due to the shift from xanthine dehydrogenase to xanthine oxidoreductase under ATP-deficient conditions during the hypoxic periods), by the mitochondrial electron transport chain, the NADPH oxidase system and unbound nitrite oxide synthase system (4,10).

The pathophysiology of ischemia-reperfusion (I/R)-induced AKI is a highly complex process that has been reported to involve the activation of neutrophils, release of reactive oxygen species (ROS) and secretion of various inflammatory mediators, including adhesion molecules (such as P-selectin and ICAM-1) and cytokines (such as TNF-α and IL-6) (13). However, there is currently no effective therapeutic option available for the treatment of renal I/R injury (RIRI), other than supportive therapies such as renal replacement therapy or hydration (14).

Lupeol is a biologically active triterpene that can be found in various edible vegetables and fruits, such as mangoes, cabbage, green peppers and strawberries (15). A previous study reported that lupeol possesses anti-inflammatory, anticancer, cardioprotective, hepatoprotective and wound-healing properties (16). Lupeol has been found to function through the toll like receptor 4/myeloid differentiation primary response 88/NF-κB p65, IL-1 receptor-associated kinase and p38 MAPK pathways (16). It has been previously tested in clinical studies for the treatment of cancer (such as bone, liver, lung, colon, rectum and bladder), actinic keratosis and nocturnal enuresis (17-19). In a study conducted with actinic keratosis patients, the birch-bark-containing Lupeol managed to clear 75% of the lesions (18). It was determined that there was a significant decrease in the number of day frequency, nocturia and total incontinence with lupeol treatment (19). Lupeol has been demonstrated to exert anti-cancer effects by promoting apoptosis, limiting cancer cell migration and invasion, decreasing cell proliferation and increasing cancer cell susceptibility to chemotherapy and radiotherapy both in vitro and in vivo (17-19).

Lupeol has been investigated in previous studies for its protective effects in an animal model of hypercholesterolemia-induced kidney damage and its effects against renal cell carcinoma in human cell culture via modulation of mitochondrial dynamics by decreased cell viability and mitochondrial fission (17,20). In rats in which hypercholesterolemia was induced by feeding a high-cholesterol diet, the decreased antioxidant status, increased renal lysosomal acid hydrolase activities and acute phase proteins, which indicates increased inflammation, were reversed by lupeol treatment (20). However, to date, to the best of our knowledge, no studies have evaluated its effects on RIRI. Therefore, the present study aimed to assess the effects of lupeol on this condition.

In the present study, it was found that renal damage occurred due to the high levels of various markers observed in the kidney tissues and blood samples from rats in group I, namely BUN, creatinine, MDA, TNF-α and caspase-3. Elevated BUN level during ischemia may be associated with tubular blockage or reverse tubular leakage. This suggests oxidative, inflammatory, biochemical and cellular damage. In addition, it was found that the levels of GSH, which is an antioxidant marker, were decreased in renal tissue following ischemia (29). Lupeol treatment was then found to exert antioxidant, anti-inflammatory and general protective effects against ischemia.

In previous studies, reperfusion was performed 30-60 min after ischemia in models of experimental RIRI (30-32). In addition, following 45 min of ischemia in rats, kidney damage commenced at 4 h and reached its maximal extent at 24 h (33). Therefore, the present study opted to terminate the ischemia procedure at 45 min followed by 24 h of reperfusion prior to euthanasia.

Renal I/R is a multifactorial process that results in a cascade of renal damage, both histopathological and functional (34). During ischemia, ROS from the xanthine oxidase system, mitochondrial electron transport chain, NADPH oxidase system and uncoupled nitrite oxide synthase (NOS) system may accumulate in ischemic cells due to low antioxidant agent concentration (10). Following tissue reperfusion, local inflammation occurs and ROS production becomes amplified, which then enters the systemic circulation to induce cell damage (renal structural damage) via apoptosis and necrosis (35). Numerous agents, such as allicin, urolithin A, empagliflozin, asiaticoside and dapsone, have all been used before and after renal ischemia to prevent this type of damage. These studies are experimental and further studies are required for clinical use (36-40).

Lupeol is a bioactive triterpene that can be found in various edible vegetables and fruits, such as mangoes, cabbage, green peppers, strawberries, olives and grapes. It can also be found in various medicinal plants, such as Bowdichia virgilioides and Crataeva nurvala (15,41,42). Previous in vivo and in vitro experimental studies have reported anti-inflammatory, antimicrobial, antioxidant, anticancer, cardioprotective, hepatoprotective, antiarthritic and wound healing effects of lupeol and its therapeutic potential (16). In the present study, lupeol demonstrated its antioxidant effects by lowering MDA levels whilst increasing those of GSH in group T compared with those in group I. To the best of our knowledge, the present study was the first in which lupeol was found to exert anti-inflammatory and antioxidant effects in a renal I/R model. Lupeol (100 mg/kg) has been previously reported to confer combined antioxidant potential and hepatoprotective effects, such as silymarin in aflatoxin B₁-induced liver injury, following the detection of oxidant (MDA and catalase), antioxidant [GSH and superoxide dismutase (SOD)] parameters and histopathological evaluation (42). In another study that previously tested the effects of lupeol for wound healing in diabetic rats, antioxidant effects were observed and stimulatory effects were exerted on wound healing (41). It has also been experimentally shown that lupeol treatment can confer antioxidant effects against acetaminophen (AAP)-induced liver injury, middle cerebral artery occlusion-induced cerebral ischemia and selenite-induced cataract and myocardial ischemia (29,43-45). In addition, antioxidant effects of lupeol have been previously demonstrated in models of liver damage, where it was observed to restore the levels of antioxidant enzymes, reduce lipid peroxidation and ROS formation, which in turn maintained the redox balance (29). Lupeol has been shown to inhibit oxidative stress-induced NF-κB activation in culture of isolated lymphocytes obtained from peripheral blood of healthy non-smoking donors (46). With neuronal cells in rat models of cerebral ischemia, lupeol treatment was found to reduce the expression levels of oxidation (by increasing SDO, GSH and decreasing ROS production) and inflammation markers (by decreasing TNF-α and IL-1β) through the activation of nuclear factor erythroid 2-related factor 2, a key antioxidant in vascular cells, through the inhibition of p38 MAPK signaling (43).

The anti-inflammatory activity of lupeol has also been investigated in the nervous system, digestive system and cardiovascular diseases. Lupeol has been documented to inhibit the expression of inflammatory genes and proteins through the TLR4/MyD88/NF-κB P65 signaling pathway, IRAK (interleukin-1 receptor-associated kinase)-mediated TLR (toll like receptor) inflammatory signaling, P38 MAPK, and JNK (c-Jun N-terminal kinase) pathways, whilst reducing that of cytokines, such as TNF-α, IFN-γ and IL-2(16). Kim et al (47) previously demonstrated in a model of cerulein-induced acute pancreatitis that lupeol treatment could reduce the degree of pancreatic edema and neutrophil infiltration, whilst inhibiting the release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6. In another study, Ahmad et al (26) reported that the optimal dose to reduce leukocyte count, IL-2, IFN-γ and TNF-α production in their cytometric investigation was 100 mg/kg of lupeol compared to 25-50-200 mg/kg. In the present study, lupeol was used to assess the possibility of these aforementioned effects in the kidney. Consistently in the present study, a significant decrease in the levels of in IL-6 and TNF-α was observed in the therapy group compared with the ischemia group.

Sudhahar et al (20) previously established a model of renal damage induced by hypercholesterolemia, where the initiation of oxidative damage in hypercholesterolemic rats was indicated by an increase in MDA levels (a product of lipid peroxidation) and a decrease in antioxidant levels (such as SOD and GSH). Additionally, Sudhahar et al (20) found that the expression of acute-phase proteins, including fibrinogen and C-reactive protein, in addition to the activity of renal lysosomal acid hydrolases (such as acid phosphatase, β-glucuronidase, β-galactosidase, N-acetyl glucosaminidase and cathepsin D), were all increased in proportion to the degree of inflammation. Subsequent histopathological findings revealed that kidney damage occurred in a hypercholesterolemic state (20), which lead to the conclusion that hypercholesterolemia increased oxidative stress and inflammation, triggering glomerulosclerosis and renal damage. By contrast, lupeol and lupeol linoleate treatment lead to the effective reversals of these aforementioned abnormalities (20).

Renal tubules can become damaged during episodes of ischemia (48). Since the inhibition of cell pyroptosis has been shown to exert a protective effect against renal I/R damage, Ni et al (48) previously measured the number of pyroptotic cells after hydrogen sulfide treatment. Decreased numbers of pyroptotic cells and reduced expression of pyroptosis protein markers, such as caspase-1, gasdermin D, IL-1β and IL-18, were observed (48). In the present study, caspase-3 expression was examined as a marker of pyroptosis, which is an indicator of inflammatory cell death (49). Caspase 3 levels were found to be lower in group T, indicating that lupeol reduced the extent of cell death. To the best of our knowledge, the only previous study on the effects of lupeol on kidney damage was the aforementioned hypercholesterolemia-induced kidney damage model, which did not examine caspase-3 expression (20). However, lupeol has been shown to inhibit caspase-3 in a rat cerebral I/R model (50).

In the present study, it was found that group I had higher BUN and creatinine levels. Increased blood levels of BUN and creatinine during ischemia may be caused by tubular blockage or reverse tubular leakage, but the exact underlying mechanism of this phenomenon remains unknown. However, acute renal failure, as indicated by increased BUN and creatinine levels, has been shown to occur before tubular necrosis develops (51). In previous studies with the effects of hydrogen sulfide and lycopene on renal I/R injury, BUN and creatinine values were found to be lower in the treatment groups (4,48). Similarly, lupeol has been shown to decrease serum BUN and creatinine values following hypercholesterolemia-induced renal damage (20). Although results from the present study did not reveal a statistically significant difference in the BUN and creatinine levels, the values were markedly decreased in group T. The reason for this improvement may be the sum of the antioxidant, anti-inflammatory and cytoprotective effects exerted by lupeol.

Previous studies have examined histological characteristics of renal I/R, such as tubular dilatation, tubular vacuolization, brush border loss, glomerular necrosis and tubular necrosis. After lycopene and hydrogen sulfide were administered to the treatment group following ischemia, a reduction in the extent of brush boundary loss, tubular vacuolization and tubular dilatation was observed (4,48). In addition, tubular epithelial denudation with casts attributable to lipemic-oxidative damage, and damage to the tubules predisposing to hypoxic injury were found in a model of hypercholesterolemia-induced renal injury, whilst intact renal architecture comparable to that of healthy kidneys was noted in the lupeol treatment groups (20). In the present study, the histopathological injury score was determined, where the highest values were found in group I. By contrast, scores close to Sham group level were obtained following lupeol treatment. The antioxidant and anti-inflammatory properties of lupeol may be partly responsible for such protective effects, similar to those previously observed in other organs, such as the eyes, brain, heart, liver and skin (16,20).

Lupeol has been shown to be effective even when applied through different routes, such as subcutaneously, topically, orally and intraperitoneally. In vivo, no systemic toxicity was observed upon treatment with 200 mg/kg intraperitoneally in rats or 2,000 mg/kg orally in mice (25). Yokoe et al (52) previously showed that subcutaneously administering lupeol can prevent local tumor progression and distant metastasis by canine oral malignant melanoma in dogs, whereas Jesus et al (53) reported that intraperitoneally administering lupeol could protect the liver and spleen against leishmaniasis. Beserra et al (41) showed that topically administering lupeol could accelerate wound healing, whilst Asha et al (44) found that orally administering lupeol alleviated selenite-induced cataracts. Another previous study, in which 200 mg/kg lupeol was administered orally to mice, found the level of lupeol in both plasma and organs such as kidney, liver, small and large intestine to be remains in high concentration and constant over time (T_1/2 for lupeol was 13.564±2.912 h) (54). Although lupeol can be found in various fruits, consumption of ≥555 g/kg mango fruit is needed to reach the dose of 100 mg/kg lupeol in humans, which was used in the present experimental study. By contrast, the oral bioavailability of lupeol was previously reported to be #x003C;1% (18). Therefore, lupeol may be more appropriately applied as a drug instead of a dietary supplement. The demonstration of antioxidant, anti-inflammatory, anticancer and anti-apoptotic effects of lupeol in both experimental in vivo and in vitro human cell cultures has supported its use for a number of human diseases. Lupeol has been tested in various clinical studies on hepatocellular cancer and actinic keratoses treatment. In addition, this drug has been previously tested a randomized placebo-controlled clinical study for the treatment of nocturnal enuresis in children (16-19,55).

In the present study, lupeol was shown to exert protective effects in the kidney against RIRI by reducing the histopathological damage score and oxidative stress, whilst suppressing the production of inflammatory proteins. In conclusion, results from the present study support the clinical use of lupeol for AKI.