Protective effect of Apigenin against folic acid-induced renal toxicity: Evaluation of body weight, kidney weight and haematological alterations

Introduction

Renal disorders have become a significant global health concern, contributing to both acute kidney injury (AKI) and chronic kidney disease (CKD), which are closely interconnected (Jiang et al., 2020) [1] . AKI is defined as a rapid and potentially reversible loss of kidney function and is commonly observed in critically ill patients (Stallons et al., 2014) [2] . Clinically, AKI is a critical condition with high rates of morbidity and mortality, affecting around 13% of hospitalized patients and contributing to nearly 1.7 million deaths annually. Additionally, it predisposes patients to CKD and end-stage renal disease (ESRD), affecting nearly 10% of the global population (Yang et al., 2025) [3] . Management of AKI remains challenging due to limited treatment options and their associated adverse effects, emphasizing the need for the development of safer therapeutic strategies (Rizwan et al., 2019 and Sadek et al., 2022) [4,5] . Folic acid (FA) is an essential, water-soluble micronutrient and an oxidized form of folate, which cannot be synthesized by humans. It serves as an important cofactor in DNA and RNA synthesis and plays a crucial role in cell growth and proliferation (Silva et al., 2017) [6] . Excessive folic acid intake has been associated with the formation of intratubular crystals, leading to impaired renal function, tubular epithelial damage, and cell death. Additionally, it promotes oxidative stress (OS) by increasing ROS generation and suppressing antioxidant enzyme activity. ROS-induced damage to lipids, proteins, and nucleic acids that triggers inflammation and contributes to the progression of AKI (Nikolic et al., 2020) [7] . Plant-based traditional medicines have been used for centuries in the treatment of various diseases. Natural products continue to serve as an important source for identifying and developing novel drugs (Fang et al., 2021) [8] . Apigenin (API), (4',5,7-trihydroxyflavone) is a naturally occurring flavone belonging to the flavonoid family, commonly found in fruits and vegetables (Fossatelli et al., 2023) [9] . Apigenin exhibits diverse biological activities, including antioxidant, anti-inflammatory, antiapoptotic, antimutagenic, and antitumor effects (Malik et al., 2017) [10] . Therefore, the present study was designed to evaluate the nephroprotective effect of apigenin against folic acidinduced AKI in mice.

Materials and Methods

Chemicals

Folic acid was procured from Sigma Aldrich, USA-(Cat. No. F7876) and Apigenin was procured from Tokyo Chemical Industry, Japan-(Cat. No. A1514).

Experimental Animals

A total of 30 adult Swiss albino mice (8-12 weeks old, 20-25 g) were obtained from Jeeva Life Sciences, Hyderabad. The animals were housed in polypropylene cages under controlled environmental conditions (temperature 22-24 °C, relative humidity 30-70%, and 12 h light/dark cycle). The mice were acclimatized for 7 days before the start of the experiment and the animals were provided with a standard pellet diet and water ad-libitum, with corncob as a bedding material. Health status was monitored daily throughout the study. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC), with approval no. (07/30/C.V.Sc, Hyd. IAEC). The animals were randomly allocated into 5 groups of six mice each (n = 6), and maintained for 14 days as per the treatment protocol given below. Folic acid control @ 250 mg/kg, single dose IP on the first day 6 III.

Apigenin control @ 20 mg/kg, IP, daily for 14 days 6

IV. Folic acid @ 250 mg/kg, single dose IP on first day + Apigenin LD @ 10 mg/kg, IP daily for 14 days 6

V. Folic acid @ 250 mg/kg, single dose IP on first day + Apigenin HD @ 20 mg/kg, IP daily for 14 days 6

Body weights

Body weight of each mouse was recorded using an electronic weighing balance (Shimadzu Pvt. Ltd.) day 0 and subsequently on days 7 and 14 of the experimental period.

Absolute kidney weights

Following sacrifice on 15 th day of experiment, the kidneys were promptly excised and gently cleared of adhering tissues. Each kidney was weighed individually using an electronic weighing balance (Shimadzu Pvt. Ltd.), and the measurements were documented.

Blood collection

At the end of the experiment (day 15), mice were fasted for 12 hours before sacrifice. Prior to euthanasia, animals were anesthetized, and blood samples were aseptically collected from the retro-orbital plexus using capillary tubes into anticoagulant-coated vacutainers (K3-EDTA tube, 13 mm x 75 mm, 6 mL; Rapid Diagnostics Pvt. Ltd., Delhi), which ensures that blood is preserved in an anticoagulated state until further analysis. The collected blood was used for the estimation of haematological parameters, including Total Erythrocyte Count (TEC), Total Leucocyte Count (TLC), Differential Leucocyte Count (DLC), Haemoglobin (Hb), and Packed Cell Volume (PCV), were analyzed using an automatic whole blood analyzer (Huma count, Med source ozone Bio-chemicals Pvt. Ltd., Faridabad, Haryana) and the results were tabulated.

Statistical analysis

Data were expressed as mean±SE (n = 6). Statistical analysis was performed using SPSS software (version 21) by one-way ANOVA, followed by Duncan's multiple range test for post hoc comparisons. A value of p<0.05 was considered statistically significant.

Results

Body weights

A significant decline (p<0.05) in body weight was observed in Group II on days 7 and 14 when compared to Group I. In contrast, apigenin-treated groups (IV and V) demonstrated a significant (p<0.05) improvement in body weight relative to Group II, indicating recovery. The body weights of Group III remained comparable to those of the control group (Group I). The findings are summarized in Table 2 and Fig. 1.

Absolute kidney weights

The absolute kidney weight in Group II was significantly (p<0.05) higher compared to the control group (Group I), indicating renal enlargement. However, treatment with apigenin in Groups IV and V resulted in a reduction in kidney weight in a dose-dependent manner, though the decrease was not statistically significant (p<0.05). Group III maintained kidney weights similar to those of Group I. Detailed results are shown in Table 3 and Fig. 2.

Haematology

Haematological analysis showed a significant (p<0.05) reduction in TEC (Fig. 3), Hb (Fig. 4), PCV (Fig. 5), lymphocytes (Fig. 6), and eosinophils (Fig. 7), along with an increase in TLC (Fig. 8), neutrophils (Fig. 9), and monocytes (Fig. 10) in group II compared to group I. Apigenin treated groups IV and V effectively reversed these changes. Whereas, no significant (p<0.05) difference was observed between groups I and III. The data are summarized in Table 4.

Discussion

Exposure to high doses of folic acid impaired the animal's physiological status, as evidenced by reduced feed intake, muscle weakness, decreased alertness, and roughening of the coat (Gupta et al., 2012) [11] . These clinical signs are indicative of folic acid-induced acute renal injury, which contributed to the observed decline in body weight. In this study, Group II animals exhibited a significant decline in body weight when compared to other groups. This reduction may be due to decreased feed intake and dehydration caused by renal damage and impaired tubular function. These findings are consistent with earlier reports by Nabae et al.

(2006) [12] ; Wan et al. (2006) [13] , and Gupta et al. (2012) [11] .

In contrast, treatment with apigenin in Groups IV and V resulted in a marked improvement in body weight when compared to Group II. The beneficial effect may be attributed to the restoration of antioxidant balance and the ability of apigenin to mitigate oxidative stress and inflammation, thereby improving overall physiological status.

The kidney index, expressed as the ratio of kidney weight to body weight, serves as a useful indicator of renal impairment. In the present study, folic acid-treated mice (group II) showed a marked increase in kidney weight compared to other groups, suggesting renal damage. This increase may be due to compensatory hypertrophy and regenerative responses following tissue damage. Earlier findings have also linked increased kidney weight associated with elevated nucleic acid synthesis (Gupta et al., 2012) [11] . These observations align with findings reported by Gupta et al. (2012) [11] and Abdelrahman et al. (2022) [14] . However, apigenin treatment in Groups IV and V resulted in a decrease in kidney weight, suggesting its protective role, possibly through anti-inflammatory mechanisms. Folic acid is essential for the synthesis of nucleic acids, amino acids, and proteins (Ratajczak et al., 2021) [15] . It also acts as a key cofactor in one-carbon metabolism required for cell growth and proliferation. Deficiency of folate impairs erythropoiesis, causing macrocytic anaemia, neurological complications such as peripheral neuropathy and increased risk of neural tube defects (NTD) in the foetus (Yan, 2021) [16] . However, high doses of folic acid can be nephrotoxic, leading to acute renal failure characterized by crystal formation in renal tubules, tubular epithelial damage, and cortical scarring (Gupta et al., 2012) [11] . In the current investigation, FA administration caused a marked (p<0.05) decline in TEC, Hb, PCV, lymphocytes and eosinophils, along with a rise in TLC, neutrophils and monocytes in group II when compared to group I. This reduction in group II haematological parameters may be attributed to impaired renal tissue producing insufficient erythropoietin (EPO), a key hormone for stimulating erythropoiesis in the bone marrow. These results are in alignment with the findings of Estrela et al. (2021) [17] and Divya et al. (2025) [18] . Haematological alterations could be attributed to enhanced oxidative stress, leading to increased RBC membrane permeability and lipid peroxidation. The significant rise in TLC, neutrophils and monocytes reflect ROS-mediated inflammation, that suggests activation of the host defense system and recruitment of inflammatory cells (Sravathi et al., 2022) [19] . In contrast, apigenin-treated groups (Group IV and V) have shown significant (p<0.05) improvement in haematological values, suggesting apigenin has beneficial role on homeostasis of hematopoietic system.

Conclusion

In conclusion, the present study revealed that folic acid (@ 250 mg/kg, IP) administration resulted in a significant alteration in body weights, kidney weights and

International Journal of Advanced Biochemistry Research https://www.biochemjournal.com haematological parameters. However, apigenin-treated groups (10 and 20 mg/kg, IP) along with folic acid (@ 250 mg/kg, IP) significantly improved these parameters, indicating its protective effects. The improvement was more pronounced at the higher dose (20 mg/kg, IP), suggesting a dose-dependent effect.

Acknowledgement

AcknowledgementsThe authors are thankful to the College of Veterinary Science, P. V. Narsimha Rao Telangana Veterinary University (PVNRTVU), Rajendranagar, Hyderabad for providing the necessary facilities to carry out the research work.

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