Rutin mitigates fluoride-induced nephrotoxicity by inhibiting ROS-mediated lysosomal membrane permeabilization and the GSDME-HMGB1 axis involved in pyroptosis and inflammation.

1. Introduction

Fluoride, a naturally occurring mineral found in geological formations, soil, and water, can be ingested and accumulate in the human body (Muthu et al., 2023). Empirical evidence suggests that moderate fluoride intake helps prevent dental caries (Jullien, 2021). Nonetheless, excessive and prolonged consumption of fluoride beyond established safety limits is associated with chronic systemic disorder development, mainly fluorosis, which causes dental and skeletal abnormalities (Kumar et al., 2023). Drinking water and food are the main sources of daily fluoride intake for most people, with drinking water making a significant contribution (Johnston and Strobel, 2020). The World Health Organization (WHO) has set a maximum permissible threshold of 1.5?mg/L for fluoride in drinking water (Guth et al., 2020). Unfortunately, over 25 countries, with a combined population of approximately 200 million people, are at risk of fluorosis owing to elevated fluoride levels in groundwater (Solanki et al., 2022). Given fluorosis has significant implications for public health and the economy, it remains a pressing global issue requiring urgent attention (Srivastava and Flora, 2020).

The kidneys function as both the primary organs for excreting waste from the body and it is a significant target for fluoride toxicity (Lash, 2019). A cross-sectional study involving 1070 adult participants revealed a substantial increase in the risk of renal impairment with elevated urinary fluoride levels (OR = 1.228; 95 % CI: 1.047, 1.439) (Wu et al., 2021). Additionally, Khandare et al. found significantly higher levels of urinary fluoride and serum creatinine in children from fluoride-affected regions than those from control regions (P < 0.05), indicating fluoride exposure as a potential risk factor for renal dysfunction (Khandare et al., 2017). Animal experiments in mice receiving 100?mg/L sodium fluoride (NaF) for 90 days revealed morphological alterations in renal tissue and impaired renal function (Wang et al., 2020). In an in vivo study conducted by Yu et al. (2022) prolonged exposure to fluoride compounds disrupted the balance between inflammation and anti-inflammation, resulting in apoptosis and inflammation in rat kidneys. Additionally, in vitro experiments have demonstrated that fluoride overexposure induces oxidative damage and apoptosis in rat proximal tubular NRK-52E cells (Gao et al., 2021). Currently, the main mechanisms underlying fluoride-induced renal injury include oxidative stress, inflammation, and cellular apoptosis (W. Li et al., 2021; Owumi et al., 2019; Wu et al., 2022). However, further research is necessary to elucidate the precise mechanisms underlying fluoride-induced renal toxicity.

Recent studies have highlighted the role of cell pyroptosis and inflammation in kidney disease development and progression (Cuevas and Pelegrin, 2021, Fu et al., 2022). Gasdermin E (GSDME), a key indicator of pyroptosis highly expressed in the kidneys, undergoes cleavage by activated caspase-3, resulting in the formation of the C-terminus (GSDME-C) and N-terminus (GSDME-N) fragments (Li et al., 2021). GSDME-N-mediated pyroptosis represents a significant mechanism of kidney tubular epithelial cell death, triggering kidney inflammation (Wang et al., 2022a). Furthermore, Li et al. found that GSDME-N-mediated renal tubular cell pyroptosis induced the release of high-mobility group box 1 (HMGB1) inflammatory factor, leading to tubular loss. Moreover, GSDME-N deficiency was observed to ameliorate renal injury by mitigating cellular pyroptosis, which led to decreased HMGB1 levels (Li et al., 2021). This suggests that the GSDME/HMGB1 signaling pathway is considered a significant mechanism underlying the pathological events of kidney injury, including cell pyroptosis and inflammation. Findings by Wen et al. demonstrated that activated caspase-3 facilitates GSDME cleavage, and inhibiting caspase-3 activation reduces damage to human kidney-2 (HK-2) cells and decreases inflammatory factor secretion (Wen et al., 2020). This indicates that cleaved caspase-3 is a significant contributor to pyroptosis. The role of cell pyroptosis and HMGB1 axis in renal injury has been elucidated; however, their specific involvement in fluoride-induced kidney injury requires further research.

Lysosomes, acting as cellular degradation centers, are pivotal in cell death processes (Mahapatra et al., 2021). Lysosomal membrane permeabilization (LMP), a key event in this process, leads to the release of cathepsin B (CTSB) and cathepsin D (CTSD) from lysosomes into the cytoplasm. This activates cytoplasmic caspase-3, thereby inducing cell apoptosis (Chang et al., 2022, Hu et al., 2022). Given that the kidney is rich in lysosomes, damaged lysosomes are implicated in kidney injury (Wang et al., 2022b). Furthermore, lysosomes serve as pivotal signaling centers that regulate cellular homeostasis (Lawrence and Zoncu, 2019). Owing to their susceptibility to various external and internal stressors, different forms of stress can induce LMP (Yang et al., 2019). Preserving lysosomal integrity and functionality is significant for maintaining cellular equilibrium. Fluorine exposure inhibits the production of intracellular free radical scavengers, which leads to a significant increase in reactive oxygen species (ROS) levels (Mohammed et al., 2017). Research conducted by Hu et al. substantiates that excessive ROS surpasses the capacity for clearance within renal cells, leading to cell apoptosis and renal damage (Hu et al., 2023). Recent evidence suggests that exposure to excessive ROS can trigger environment- and cell-type-specific LMP, resulting in cell death and inflammation (Cai et al., 2018). Nonetheless, it remains unclear whether fluoride-induced ROS orchestrates LMP and subsequent renal damage. Therefore, additional studies are warranted to elucidate this intricate interaction.

Rutin, a polyphenolic flavonoid found naturally in various fruits and vegetables, is known for its robust antioxidant and anti-inflammatory properties (Muvhulawa et al., 2022). Studies have shown that rutin can achieve these effects through multiple pathways, including neutralizing intracellular free radicals, preventing further oxidation of free radicals, enhancing intracellular antioxidant enzyme activities, and inhibiting HMGB1 expression and release (Arab et al., 2022, Chen et al., 2022; T. Liu et al., 2022). For example, Khan et al. conducted a trial demonstrating the hepatoprotective properties of rutin against carbon tetrachloride (CCL4)-induced hepatotoxicity in rats by increasing endogenous hepatic antioxidant enzymes (Khan et al., 2022). This finding suggests that the therapeutic mechanism of rutin may involve enhancing intracellular antioxidant responses, reducing intracellular ROS production and accumulation, and mitigating cellular inflammation. Furthermore, rutin mitigates cellular inflammatory responses by inhibiting the expression of markers associated with focal death (Yi, 2018). Rutin demonstrates protective effects in other metabolic disorders, such as diabetic nephropathy and neurodegenerative diseases; however, its underlying mechanism in fluoride-induced renal injury remains unclear (Enogieru et al., 2018, Ghorbani, 2017). Additionally, research investigating the influence of rutin on lysosomal membrane permeabilization and the expression of the GSDME/HMGB1 axis, which are implicated in renal cellular pyroptosis and inflammation under fluoride-exposed conditions, is limited.

Therefore, this study aims to investigate the influence of fluoride on renal damage. Moreover, it aims to elucidate whether NaF can initiate renal cell pyroptosis and inflammation through ROS-mediated LMP and GSDME/HMGB1 and clarify the role of rutin in this process. Our findings revealed that NaF induces ROS accumulation by influencing intracellular free radical antioxidant enzyme activity. This process triggers lysosomal membrane permeabilization, ultimately leading to renal cell pyroptosis and inflammation. Further, we discovered that rutin significantly alleviates ROS generation and accumulation. Intervention with rutin markedly improved LMP and inhibited the GSDME/HMGB1 pathway, thereby ameliorating NaF-induced renal injury. Owing to its natural antioxidant and anti-inflammatory properties, rutin can be utilized as a dietary supplement, offering a novel approach to prevent and manage fluoride-induced nephrotoxicity.

3. Results

4. Discussion

This study builds upon earlier research into fluoride-induced renal damage. It specifically shows that fluoride increases ROS levels, leading to renal cell pyroptosis and inflammation through LMP and the GSDME/HMGB1 pathway. Moreover, our findings revealed that rutin could alleviate NaF-induced renal injury by mitigating oxidative damage and modulating the activation process of the LMP and GSDME/HMGB1 axis.

The significance of cellular pyroptosis and inflammation in renal injury is well established. In this study, we found a significant increase in the expression levels of key indicators of pyroptosis, including Cleaved Caspase3 and GSDME-N, and the inflammation factor HMGB1 in vivo and in vitro. GSDME-N, identified as a key executor of pyroptosis, indicates the onset of this process when its levels rise (Wei et al., 2023). When cellular pyroptosis or necrosis occurs, HMGB1, an essential mediator secreted by immune cells, is released extracellularly, thereby initiating sterile inflammation by activating various receptors through multiple pathways (Tan et al., 2020). Previous studies conducted by Xia et al. and Shen et al. found that the cleavage and activation of GSDME promote the release of HMGB1 in cisplatin-induced renal injury, exacerbating renal cell pyroptosis and inflammation, thereby worsening the nephrotoxic effects of chemotherapy drugs (X. Shen et al., 2021; Xia et al., 2021). Our findings demonstrate that renal cell pyroptosis and inflammation occur in the kidneys of offspring rats exposed to 100?mg/L NaF for 60 postnatal days and in HK-2 cells exposed to 60?mg/L NaF for 24?h compared to the presented data. Furthermore, the proportions of Cleaved PARP1 were significantly upregulated. The research conducted by Tian et al. reported that the administration of 100?mg/L NaF for 90 postnatal days resulted in vacuolar degeneration, lysosome enlargement, and apoptosis in renal tubular epithelial cells of SD offspring rats. This finding supports our assertion that prenatal and lactational exposure to fluoride could lead to renal injury in adulthood (Tian et al., 2019). Significantly, these findings are consistent with epidemiological study findings, indicating that children and adolescents are more susceptible to the adverse influence of fluoride poisoning (Yadav et al., 2019). In summary, our study provides evidence that prolonged exposure to fluoride stimulates renal injury, potentially through activation of the GSDME/HMGB1 signaling pathway. This validates our successful establishment of a fluoride-induced renal toxicity model in rats and a NaF-intoxicated HK-2 cell model.

Lysosomal integrity plays a significant role in facilitating cellular adaptation to various signals and stimuli (Yang and Wang, 2021). In our study, we observed an increase in the expression of lysosomal cathepsins, CTSB and CTSD, both in vivo and in vitro, following NaF exposure. These enzymes, CTSD and CTSB, are the principal hydrolytic enzymes situated within lysosomes, responsible for degrading several substrates. Loss of lysosomal membrane integrity and increased permeability can lead to the release of CTSB and CTSD into the cytosol, subsequently triggering multiple cell death pathways, such as apoptosis, pyroptosis, necrosis, and ferroptosis (Zhu et al., 2020). Furthermore, we discovered that transmission electron microscopy and AO fluorescence staining revealed evidence of LMP in HK-2 cells. A recent study conducted by Wang et al. (2022b) reported similar findings, demonstrating that CTSB release subsequent to LMP induced HK-2 cell apoptosis. Conversely, exposure to 60?mg/L NaF resulted in decreased expression and activity of cathepsin in neuronal cells. These disparities can be attributed to variations in the assessed individuals, target organs, and actual exposure doses (Zhang et al., 2023). Furthermore, research by Chen et al. indicated that oxidative stress promoted LMP and cathepsin release in neurons, resulting in cellular pyroptosis (Chen et al., 2019). Numerous corroborative studies have collectively supported the concept that low levels of ROS regulate cell survival signaling, while excessive ROS can induce oxidative stress, ultimately leading to cell death (Y. Liu et al., 2022; Yang and Lian, 2020). Our research findings demonstrated that NaF induces a significant, dose-dependent increase in intracellular ROS levels. Liu et al. provided evidence suggesting that ROS precedes lysosomal damage in the context of macrophage pyroptosis and inflammation induced by iron oxide nanoparticles. They found that the ROS inhibitor NAC significantly reduced lysosomal injury. In contrast, the CTSB inhibitor had no statistically significant difference in ROS production (Liu et al., 2018). Consequently, we hypothesize that fluoride exposure-mediated ROS contribute to LMP.

NAC, known for its ability to inhibit ROS, has been shown to mitigate cell injury by reducing oxidative stress and inflammatory response, thereby improving kidney diseases (Y. Shen et al., 2021). Consistent with this, our findings suggest that co-treatment with NAC and NaF led to a significant decrease in ROS levels. Furthermore, NAC intervention effectively reduced the expression levels of NaF-induced CTSB and CTSD proteins, thereby improving LMP. Subsequently, we found that Cleaved Caspase3, GSDME-N, Cleaved PARP1, and HMGB1 upregulation induced via NaF was inhibited following NAC treatment. Zhang et al. found that NAC maintains cell membrane stability by inhibiting and improving ROS levels and LMP, respectively (Zhang et al., 2012). Further, X. Shen et al. (2021) reported that NAC suppresses intracellular ROS accumulation and inhibits pyroptosis by suppressing the phosphorylation of JNK and downregulating the expression levels of Cleaved Caspase3 and GSDME-N. Additionally, studies conducted by Singh-Mallah et al. revealed that NAC reduces the release of HMGB1 (Singh-Mallah et al., 2021). In summary, our research findings confirm that NAC can inhibit NaF-induced ROS level elevation. This suggests that NaF contributes to renal injury by inducing ROS generation, which mediates LMP and activates the GSDME/HMGB1 axis. This activation leads to renal cellular pyroptosis and inflammation.

Rutin, a naturally occurring flavonoid, has been extensively researched and shown to mitigate inflammation by regulating oxidative stress (Hu et al., 2021). Significantly, its role aligns with that of NAC. By suppressing oxidative stress, it restores LMP, improves pyroptosis, decreases inflammation, and ultimately alleviates renal injury in this study. Undeniably, multiple mechanisms may drive this process, such as luteolin, a flavonoid, which attenuates renal inflammation and necrotic cell apoptosis by attenuating the NF-?B p65 and MAPK signal cascades (Ren et al., 2020). Furthermore, though rutin is considered beneficial for rats exposed to fluoride, no developmental effects were observed when C57BL/6?J female mice consumed substantial amounts of rutin during gestation and lactation (Lesser et al., 2015, Oyagbemi et al., 2018). The difference observed could be attributed to factors such as animal species, strain, and physiological status. Owing to the limited size of the animal sample, we did not perform further rutin interventions in the offspring rats after fluoride exposure to understand the potential long-term effects of rutin fully. However, our findings provide insight into the mechanism underlying the beneficial role of rutin in fluoride-induced renal injury. Inferred from the findings of previous research, we found that rutin achieves renal protection against fluoride by modulating the ROS-mediated LMP and regulating the expression of the GSDME/HMGB1 axis. This modulation helps prevent the onset and progression of fluoride-induced nephrotoxicity.

5. Conclusion

Our in vivo and in vitro experimental findings confirm that excessive fluoride intake induces LMP and activates the GSDME/HMGB1 pathway by stimulating oxidative stress, resulting in pyroptosis, inflammation, and renal injury. Significantly, we have identified rutin as an intervention agent capable of not only reducing ROS production effectively but also mitigating LMP and inhibiting the expression of GSDME/HMGB1. This intervention improves renal cell pyroptosis and inflammation, thereby treating fluoride-induced renal injuries. Our research findings provide a novel therapeutic approach for mitigating fluoride exposure-induced nephrotoxicity, establishing a theoretical foundation for the application of rutin in preventing and treating fluorosis.

Funding

This study was supported by grants from the National Natural Science Foundation of China (Grant Nos. 82360671 and 82060580), the Bingtuan Program of Science and Technology Innovation (Grant No. 2021CB046), as well as the Shihezi University International Science and Technology Cooperation Promotion Programme Project ( No. GJHZ202308).

CRediT authorship contribution statement

Li Liu: Visualization. Xueman Ding: Visualization. Panpan Xu: Writing – review & editing. Qiang Niu: Resources, Funding acquisition, Conceptualization. Yue Ma: Writing – original draft, Investigation, Formal analysis. Tingting Li: Methodology. Yue Zhang: Data curation. Hengrui Xing: Writing – review & editing.