Resveratrol Attenuates Arsenic Trioxide-Induced Nephrotoxicity in Mice: A Histological Study

Introduction

Arsenic trioxide (ATO, As₂O₃), is a potent anti-neoplastic agent used to treat acute promyelocytic leukemia (APL).^1,2 ATO is the drug of choice for APL, but there is no established treatment for preventing arsenic-mediated toxicity in other organs during chemotherapy. It has been reported that patients treated for APL suffer from nephrotoxicity caused by ATO, thus making it unsafe.³ Nephrotoxicity manifests as wide-spectrum damage to different segments of the nephron, depending on the mechanism of action of the drug administered.⁴ Arsenic (As) is metabolized by a process called biomethylation, which takes place in the cells lining the proximal convoluted tubules (PCTs) of the kidney cortex and in the liver cells, making these the target sites for As-induced toxicity.⁵ Biomethylation is a process of the addition of one or more methyl groups (-CH₃) to a chemical species. Glutathione (GSH), an intracellular antioxidant, plays a major role in biomethylation.^6,7 Due to chronic arsenic exposure, trivalent arsenic binds to the thiol group of GSH and makes an As-glutathione complex, i.e., arsenic Tri-glutathione or As^III (GS)₃. This complex, along with the intermediate metabolites formed in arsenic biomethylation, inhibits the enzyme glutathione reductase (GR), required to regenerate GSH.^7,8 This leads to decreased levels of GSH and increased oxidative stress, which damages PCT cells, and this leads to acute tubular injury (ATI). GSH levels may be maintained by extrinsic antioxidants.

Resveratrol (Res) is synthesized by plants in response to fungal infection, mechanical injury, or radiation. It is abundant in wine grapes (Vitis vinifera), peanuts, pistachios, soybeans, dark chocolate, and cranberries, and has been proven to have many beneficial effects, including anti-cancer, antioxidant, anti-microbial, and anti-inflammatory properties. Res can maintain GSH levels and has shown promising effects in preventing ATO-induced cardiotoxicity⁹ and hepatotoxicity in mice.¹⁰ However, there is a paucity of histological studies with sufficient sample size on its effect on ATO-induced nephrotoxicity.

We investigated the effect of Res on renal histology in a model of ATO-induced nephrotoxicity in mice. This may have implications in preventing the As-induced toxicity in regions that are endemic to high levels of this metal in the groundwater.

Materials and Methods

Results

Histopathology

The histological results were similar with both H&E- and PAS-stained sections obtained from the same animal. The basement membrane and microvilli (brush border) were best seen in PAS-stained sections [Figures 1a and b], since PAS stains the basement membrane and brush borders with a different (magenta) color for better differentiation [Figures 1c and d].

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Figure 1:

Histology of normal mouse kidney. (a) H&E-stained (100x), arrows point to PCT (lumen not seen) & DCT (visible lumen), (b) H&E-stained (400x), arrows indicate tabularization of parietal layer of Bowman’s Capsule, (c) PAS-stained (100x), black line at cortico-medullary junction, and (d) PAS-stained (400x), basement membrane and brush border distinct (bright magenta color). G: Glomerulus, BV: Blood vessel, PCT: Proximal convoluted tubule, DCT: Distal convoluted tubule, H&E: Hematoxylin and eosin.

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Sections from Group I showed clearly distinct cortex containing glomeruli and tubules (proximal & distal convoluted), as well as medulla comprising predominantly of tubules (straight segments, loops of Henle & collecting). Within the cortex, the tubules were observed to be closely packed. Epithelial cells lining the proximal convoluted tubules (PCTs) stained intensely and showed euchromatic round nuclei, abundant cytoplasm, and intact brush borders on their luminal surfaces. The distal convoluted tubules (DCTs) were lined by smaller, lightly stained cells with sub-apical nuclei and basal striations. The lumina of PCTs were difficult to discern, while the DCTs showed well-defined lumina [Figure 1a]. The glomeruli appeared normal in all cases, with intact glomerular basement membranes. The Bowman’s capsule of a few glomeruli showed tubularization, i.e., simple squamous epithelium was replaced by simple cuboidal epithelium in the parietal layer [Figure 1b].

Groups I, II, and IV showed mild to moderate edema, while ATO-treated Groups III and V showed marked interstitial edema, which was identified by an increased distance between the tubules [Figures 2 and 3]. Moderate to severe ATI was observed in all eight cases of Group III with focal to diffuse involvement of the cortical area, indicating the causative role of arsenic in the pathogenesis of ATI. The features of tubular damage that were observed included dilation of the PCT lumen and flattening of the tubular lining epithelium due to complete loss of the brush border, blebbing, and sloughing of cells into the lumen [Figure 2]. Mild to moderate ATI was seen in a few sections from Groups I, II, IV, and V, whereas moderate to severe ATI was observed in all sections of Group III.

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Figure 2:

Histology of mouse kidney cortex (Group III), showing acute tubular injury (ATI). (a) H&E stain, (400x) and (b) PAS-stain (400x). Increased inter-tubular distance indicates interstitial edema, (*) indicates dilation of PCT lumen due to loss of brush border, black arrows indicate blebbing and sloughing of cells into lumen.

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Figure 3:

Histology of mouse kidney cortex (Group V), showing marked interstitial edema. (a) Periodic acid Schiff (PAS)-stain (100x) and (b) PAS-stain (400x). (*) Marks increased inter-tubular distance, but tubules exhibit normal histology.

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Interstitial inflammation was observed in all groups, with no significant differences in inflammation scores (p > 0.99). Lymphocytes and plasma cells were seen in the interstitial infiltrate. Apart from interstitial inflammation, random cases showed inflammation of the renal pelvis, indicating pyelonephritis. Vacuolation of the PCT lining cells was observed in Groups II, III, IV, and V [Figure 4].

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Figure 4:

Histology of mouse kidney cortex (Group V), showing vacuolation in PCTs. (a) H&E-stain (400x) and (b) PAS-stain (400x), black arrows point towards vacuoles in lining epithelial cells of PCT. BV: Blood vessel, PCT: Proximal convoluted tubule, DCT: Distal convoluted tubule.

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Statistical analysis

The ATI and TCI scores were significantly higher in Group III [Figure 5] than in Group V (p < 0.05, Table 1), suggesting a role for res in attenuating ATO-induced renal injury. The ATI score of Group III was significantly higher than that of Group I (p = 0.002), Group II (p = 0.003), Group IV (p = 0.001), and Group V (p = 0.017). TCI score was also significantly higher in Group III [Figure 5] in comparison with Group I (p = 0.01), Group II (p = 0.001), Group IV (p = 0.002), and Group V (p = 0.028).

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Figure 5:

Bar diagram representing interstitial edema, interstitial inflammation, acute tubular injury, and total cortical injury scores as mean ± SD. (*) indicates p <0.05, (**) indicate p <0.01. NC: Normal control, VC: Vehicle control, ATO: Arsenic trioxide treated (2 mg/kg of body weight), Res: Resveratrol treated (40 mg/kg body weight), and ATO+Res treated (2 mg/kg body weight) followed by resveratrol (40 mg/kg body weight) after 1 h.

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Table 1: Statistical analysis of histo-pathological scores

Group (n = 8)	Experimental protocol	Interstitial edema	Interstitial inflammation	ATI score	TCI score
I	Normal control	1.50 ± 0.53	1.25 ± 0.46	0.63 ± 1.06	0.563 ±1.39
II	Vehicle control	1.13 ± 0.64	1.13 ± 0.83	0.62 ± 0.91	0.250 ± 0.70
III	ATO-treated	2.13 ± 0.83	1.38 ± 0.74	2.25 ± 0.70	2.438 ± 1.39
IV	Res-treated	1.13 ± 0.83	1.13 ± 0.35	0.50 ± 0.75	0.375 ± 0.69
V	ATO + Res	1.63 ±1.18	1.38 ± 0.74	0.87 ± 0.83	0.688 ± 0.84

Data are expressed as mean ± SD. Group comparison was analyzed using the Kruskal-Wallis test. p <0.05 was considered statistically significant. Interstitial edema p values (Group II vs. Group III = 0.025, Group III vs. Group IV = 0.016); Interstitial inflammation (p >0.99 all groups); ATI p values (Group I vs. Group III = 0.002, Group II vs. Group III = 0.003, Group III vs. Group IV = 0.001, Group III vs. Group V = 0.017); TCI p values (Group I vs. Group III = 0.01, Group II vs. Group III = 0.001, Group IV vs. Group III = 0.002, Group III vs. Group V = 0.028).

Interstitial edema scores were significantly higher in Group III than Group II (p = 0.025) and Group IV (p = 0.016). There were no significant differences [Figure 5] in interstitial inflammation scores.

Discussion

In this study on Swiss albino mice, we found that Res attenuates the histo-pathological damage to the kidneys induced by ATO. Mice treated with ATO alone exhibited skin lesions, including patchy hair loss and skin ulceration. This is a common finding usually reported as patchy hair loss.¹⁸ Histological examination revealed tubularization of the parietal layer of Bowman’s capsule in renal corpuscles of the cortex, uniformly in all groups. This has been reported as physiological with increasing age.¹⁹ In humans, however, glomerular tubularization is an important sign of renal injury.

Compared with the controls, ATO-treated Groups III and V showed marked interstitial edema. These observations are consistent with those of Adil et al. (2005), who reported renal damage in adult male Sprague-Dawley rats following 28 days of sodium arsenite administration, manifesting histologically as edema and inflammatory infiltration.²⁰ Interstitial inflammation was seen in murine renal tissues obtained from all groups, as corroborated by Duan et al. (2022), who reported that arsenic induces a continuous immune-inflammatory response in the kidney.²¹ Inflammation of the renal pelvis lining epithelium suggests pyelonephritis; possible causes include poor hygiene (soiled bedding with urine/feces) and lack of antibiotic supplementation at the Animal Facility. Vacuolation observed in PCT lining cells has been described as a normal feature of the mouse kidney with advancing age (increased vacuolation observed in an age-dependent manner in 4-9-month-old mice), especially in male mice.^19,22 Male-specific occurrence of these vacuoles suggests that these are directly/indirectly related to androgens. Boundaries of these vacuoles were found to be enriched with a lysosomal marker, LAMP-1 (lysosome associated membrane protein 1), suggesting their lysosomal origin.²² In humans, however, vacuolation is a common feature of degeneration mostly seen in PCTs, indicating drug-induced nephrotoxicity.^22,23 Their role in physiology and pathology has not yet been studied, warranting additional studies to clarify the functional role of these vacuoles and their contribution to pathological processes and ageing.

All the features of ATI (blebbing, sloughing, and brush border loss leading to dilated lumen) were found to be more pronounced in renal tissues obtained from Group III mice, as compared with those from other groups, especially in comparison to Group V (ATO+Res). In this study, 3 out of 8 normal control mice showed mild ATI, which may be ascribed to perfusion-related changes occurring during euthanasia. Significantly higher TCI scores (indicating a greater percentage area of cortical damage) were found in renal tissues of Group III mice, compared with those of other groups, including Group V (ATO+Res). This indicates that Res has the potential to protect against renal injury induced by exposure to Arsenic.

These results are further corroborated by similar findings of Yu et al. (2013),⁹ who injected Chinese Dragon-Li cats with 1 mg/Kg ATO on alternate days, preceded by 3 mg/Kg Res via the forearm vein 1 h before the ATO treatment. They concluded that Res protects against ATO-induced nephrotoxicity in cats. The probable mechanism for this protective effect is that Res maintains the GSH level in cells,²⁴ and therefore aids arsenic biomethylation by converting naturally occurring toxic As to its less toxic forms, which are readily excreted through urine.²⁵ As a previous study has reported, some patients with APL develop kidney injury following ATO treatment.²⁶ Duration of ATO treatment (at dose 0.15 mg/kg body weight) varies patient to patient, and treatment is continued until complete remission is achieved.²⁷ Kidneys are involved in the biomethylation of arsenic and therefore are the worst-affected. The major mechanism behind arsenic-induced kidney injury is oxidative stress.²⁸

Arsenic is naturally present in water bodies and soil in many countries, including India, China, Pakistan, and Bangladesh.²⁹ In India, the states of West Bengal, Assam, Bihar, Jharkhand, and Uttar Pradesh are among the most severely affected.³⁰ More than 50 million people are said to be affected by the high levels of arsenic in drinking water along the Ganga and Brahmaputra basins.³¹ Several studies have reported an association between chronic arsenic exposure through contaminated drinking water and the presence of proteinuria among populations residing in high-arsenic areas.^32,33 Given that both therapeutic and environmental arsenic exposure can induce renal injury and arsenic mainly induces damage through oxidative stress-mediated mechanisms, the present findings suggest that Res-based interventions may hold promise in mitigating arsenic-related nephrotoxicity in patients with APL as well as in populations exposed to contaminated drinking water.

This study has a few limitations – it was designed as a purely histological evaluation and therefore focused on structural alterations within the kidney tissue. While H&E and PAS staining provide a comprehensive overview of tissue architecture and pathological changes, they do not allow detailed analysis of cellular pathways, oxidative stress, or kidney function. Another key limitation is that renal functional parameters (urea, creatinine, and urine protein) were not measured. Therefore, the functional impact of arsenic exposure and the potential protective effects of Res on renal function remain unverified. Studies integrating molecular markers (especially oxidative stress markers) and renal function tests would help to further expand upon these observations.

In conclusion, we compared histological data from kidneys of control, ATO,and Res-treated groups using standardized histopathological scoring criteria (ATI & TCI scores) to demonstrate the protective effect of Res on renal cortex damaged by exposure to Arsenic. The results suggest that Res (administered post-exposure) has the potential to reverse ATO-induced renal injury. Although further quantitative and biochemical studies are required for corroboration, these results may be extrapolated for future therapeutic application to mitigate the deleterious effects of As toxicity in arsenic endemic areas.