Zinc (Zn) is an essential trace element, which is involved in many important physiological processes (Hutchens et al., 2021). In eukaryotes, Zn plays an important role in biological processes such as energy metabolism, oxidative stress and signal transduction (Kim and Lee, 2021). A number of studies have shown that appropriate amount of Zn added to feed can improve the growth efficiency of animals (Li et al., 2021c). However, excessive intake of Zn can cause acute and chronic poisoning in animals (Kataba et al., 2021). Studies have pointed out that the toxic effect of Zn is mainly through disrupting the physiological homeostasis of lipid bimolecular structure and changing the structure of glycoprotein on the cell surface, and causing disorders to the structure and function of Zn-containing enzymes such as ATP and nucleotidase, resulting in reduction of ATP synthesis, dysfunction of membrane active transport, organelle edema and other diseases (Alhasawi et al., 2014; Ren et al., 2017). When animals are supplemented with excessive Zn, they will show loss of appetite, sluggish activity, diarrhea, growth and development arrest and other toxic symptoms. Recently, lots of studies have shown that the homeostasis of Zn is related to the occurrence and development of various diseases (Wang et al., 2020).

Hesperidin is a derivative of dihydroflavone and mainly exists in the peel of citrus fruits (Xiong et al., 2019). In recent years, hesperidin has attracted extensive attention in livestock and poultry breeding because of its antioxidant, lipid metabolism regulation, anti-inflammatory and other biological activities (Hager-Theodorides et al., 2021). It has been reported that hesperidin can effectively relieve the symptoms of heat stress in broilers and improve the growth efficiency of the animals, so it is gradually recommended to be used in animal feed as a feed additive (Kamboh et al., 2013; Kouvedaki et al., 2024). Studies have found that hesperidin regulates gut flora to alleviate liver damage in mice (Li et al., 2022). However, there are few studies on the mechanism of hesperidin in toxic diseases and in the regulation of intestinal flora.

The “gut microbiota-gut-kidney axis” theory was first proposed to explain how changes in gut microecology affect chronic kidney disease through regulation of metabolites (Nouri et al., 2022). Recent studies have found that a decrease in the abundance of intestinal probiotics in animal models of chronic kidney disease, accompanied by an increase in the abundance of pathogenic bacteria, which causes dyshomeostasis of intestinal flora, directly destroys intestinal barrier function and leads to bacterial translocation and the accumulation of enterogenous uremic toxins in the blood, thereby activating renal inflammation, oxidative stress and fibrosis pathways (Plata et al., 2019; Li et al., 2023a). In addition, excessive metabolic waste cannot be fully excreted by the kidney and re-enters the intestine, further aggravating the imbalance of intestinal flora and leading to the sustainable development of the disease. Therefore, the discovery or development of effective drugs through the “gut microbiota - enteric-kidney axis” is of great significance for the treatment of kidney diseases (Hsu and Tain, 2022). At present, there are still gaps in the research on the role and mechanism of “gut - kidney axis” in natural compounds on chronic kidney injury caused by heavy metals in animals, which is also the focus of the scientific community in recent years.

Studies have confirmed that high levels of Zn can induce kidney damage (Ratn et al., 2018). At the same time, hesperidin has been shown to have significant effects in maintaining intestinal flora homeostasis and alleviating kidney injury, but its underlying mechanism is still unclear (Mas-Capdevila et al., 2020). In this study, pigs were used as experimental animals, and hesperidin was used for treatment based on the establishment of a pathological injury model of high levels of Zn. Based on the theory of “intestinal microbiota - enteric-kidney axis”, changes in intestinal flora structure and metabolites, changes in intestinal barrier function, renal function, and dynamic processes of autophagy and apoptosis were detected by detecting changes in intestinal flora of pigs. Further, the mechanism of “intestinal flora - intestinal renal axis” in hesperidin alleviating kidney injury caused by Zn poisoning was discussed, which laid a foundation for elucidation of the protective mechanism of hesperidin in regulating kidney injury of pigs with high levels of Zn.

Zn is an essential trace element involved in a variety of life processes. Dietary supplementation of appropriate amount of Zn can not only improve animal reproductive performance and maintain intestinal microenvironment homeostasis, but also improve antioxidant function and enhance immunity. However, excessive intake of Zn in animals can lead to decreased antioxidant function, resulting in oxidative stress and programmed death (Yang et al., 2022; Li et al., 2023b). It has been proved that the kidney is an important target organ for Zn toxicity, but its pathogenic mechanism is still unclear. The “gut-kidney axis” theory holds that the disturbance of intestinal flora can induce the impairment of intestinal barrier function and systemic micro-inflammatory response, thus inducing the functional impairment of the kidney (Yang et al., 2018). Therefore, through the search for corresponding drugs, through the “entero-renal axis” to treat kidney disease has become a hot research direction. Hesperidin is a kind of natural flavonoid widely found in citrus fruits, which has been found to have antioxidant, anti-inflammatory and other biological activities (Li et al., 2021b). Here, this study identified the mechanism of “intestinal flora - intestinal kidney axis” in hesperidin alleviating kidney injury caused by Zn, and further clarified the pathway of hesperidin in Zn-induced kidney injury.

A large number of studies have confirmed that cell damage caused by heavy metals is often accompanied by autophagy and apoptosis (Fang et al., 2021; Li et al., 2021a). Autophagy is a biological mechanism widely existing in eukaryotic cells, and it is considered to be an important way for cells to degrade large quantities of senescent proteins and damaged organelles. Autophagy forms an independent double-layer membrane structure, wraps the cytoplasm under the regulation of a variety of proteins, fuses with lysosomes and finally degrades into small molecules, which are released back into the cytoplasm for use (Klionsky et al., 2021). During autophagy, ATG5 promotes the formation of bilayer membrane of autophagosome, recruits LC3 and promotes its transformation from LC3-I to LC3-II. The degradation of p62 is another marker for monitoring autophagy, as p62 can bind to LC3 and be selectively degraded by autophagy (Levine and Kroemer, 2019). In addition, apoptosis is a type of programmed cell death that is highly regulated by multiple genes and characterized by chromatin condensation, DNA cleavage, and the formation of apoptotic bodies. The mechanism of apoptosis is very complex, mainly divided into endogenous and exogenous pathways (D'Arcy, 2019). Among them, most of the research focuses on endogenous apoptosis, and the apoptosis of mitochondrial pathway has also become a research hotspot for most scholars. In most cases, mitochondria-mediated endogenous apoptosis is controlled by the Bcl family, which is composed of pro-apoptotic factors such as Bax and Bak-1 and anti-apoptotic factors Bcl-2 (Yuan et al., 2021). When the apoptotic signal is received, the pro-apoptotic protein will be transferred to the mitochondrial membrane, and the Bcl-2 protein will be further down-regulated, resulting in the permeability of the mitochondrial outer membrane, the reduction of mitochondrial membrane potential, and the release of intermembrane pro-apoptotic substances, which will further promote the shear of Caspase-3, destroy the nuclear structure, and break down the cell into apoptotic bodies (Liao et al., 2019). It has been confirmed that feeding high dose of copper can increase the protein expression levels of LC3-II/LC3-I, ATG5, Bax, and cleaved Caspase-3 (Liao et al., 2020, 2021). Zhang et al. also confirmed that molybdenum and cadmium combined feeding in ducks also led to renal autophagy and apoptosis (Zhang et al., 2022b). In our study, we have found that Zn could induced the high expression level of autophagy and apoptosis-related genes, and hesperidin could reverse these effects, which verified the anti-renal damage effect of hesperidin.

Studies have shown that the toxic effect of heavy metals is caused by the absorption of digestive tract and metabolism in the body (Witkowska et al., 2021). At the same time, intestinal damage may become an important bridge for toxin-induced organ toxicity damage (Liao et al., 2022). Therefore, the evaluation of intestinal damage is also an important indicator for heavy metal poisoning. In this study, we evaluated the intestinal toxicity of Zn by histology and expression of ZO-1 protein. ZO-1 is one of intestinal tight junction proteins, which can maintain the structural integrity of intestinal epithelial cells and the continuity of intestinal mucosal barrier, and plays an important role in cell proliferation, differentiation and growth, regulation of intercellular signal transduction and penetration (Kuo et al., 2022). Here, we found that Zn exposure could cause pathological damage to the colon, and induce the ZO-1 protein expression decreased significantly. Nevertheless, hesperidin treatment could upregulate the ZO-1 expression level, which indicating that hesperidin can repair barrier barriers, improve the structure and function of intestinal epithelium, and regulate intestinal mucosal permeability.

In this study, we have revealed that Zn can cause nephrotoxicity, and the colon also produces pathological damage, and hesperidin can reverse the damage both of them. It has reported that the process of drug alleviating kidney injury may involve the role of the “gut-kidney” axis, especially the function of intestinal flora (Xie et al., 2022). Therefore, we speculate that hesperidin plays a key role in alleviating Zn-induced nephrotoxicity by the gut microbiota. In recent years, with the attention paid to the occurrence of kidney diseases caused by intestinal microecological disorders, the relationship between gut and kidney has gradually become a hot topic of research, and the theory of “gut-kidney axis” has been gradually confirmed (Evenepoel et al., 2017). As reported, 20% of patients with inflammatory bowel disease have mild tubular damage, which may be related to the effects of conventional treatment drugs and inflammatory cytokines. The proposal of the “gut-kidney” axis explores the regulating effect of the gut on the kidney. As an important role of the “gut-kidney” axis, intestinal flora plays an important role, and the disturbance of gut microbiota homeostasis and the intestinal barrier dysfunction are the inducing factors in the occurrence of kidney diseases (Chen et al., 2019). Here, we found that Zn could remarkably increase the abundance of Tenericutes and Christensenella, and hesperidin significantly decrease their abundance. Zhang et al. showed that the Tenericutes was reduced in diabetic nephropathy after Lycoperoside H treatment, which provided an important reference for the involvement of Tenericutes in the regulation of kidney diseases through “gut-kidney” axis (Zhang et al., 2022a). In addition, as a probiotic, Christensenella has been used for the treatment of diabetes, which could enhance the intestinal barrier and reduce intestinal inflammation (Pan et al., 2022). Therefore, hesperidin was found to improve Zn-induced intestinal barrier dysfunction by changing the abundance of Tenericutes and Christensenella, thus alleviating zinc-induced kidney injury via “gut-kidney” axis. In subsequent studies, we can use high-throughput sequencing to screen signal molecules to explore the molecular targets of hesperidin in regulating zinc-induced kidney injury.

In summary, Zn could induce nephrotoxicity and intestinal damage, and the disordered intestinal flora, as the core of “gut-kidney” axis, is an important pathway to promote Zn-induced nephrotoxicity. Additionally, hesperidin could improve the zinc-induced gut microbiota disorder for alleviating Zn-induced nephrotoxicity via “gut-kidney” axis.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal studies were approved by South China Agricultural University ethics committee. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

QY: Writing – original draft. LQ: Writing – review & editing. SH: Writing – review & editing. CZ: Writing – review & editing.