Baicalin modulates endoplasmic reticulum-Golgi homeostasis via targeting ATF6-GBF1 axis to alleviate copper-induced nephrotoxicity in duck

Abstract

Copper (Cu) exposure caused by environmental contamination or dietary imbalance poses a potential threat to poultry health by inducing renal dysfunction and disrupting cellular homeostasis. Baicalin (BA), a natural flavonoid with antioxidant and anti-inflammatory properties, has shown renoprotective potential. However, whether and how BA alleviates Cu-induced nephrotoxicity remains unclear. Disruption of endoplasmic reticulum (ER)-Golgi trafficking impairs cellular homeostasis and may contribute to renal injury, but its role in Cu-induced kidney damage has not been fully defined. In this study, using duck in vivo and in vitro Cu exposure models, we found that Cu downregulated the Golgi structural protein GBF1 and disrupted ER-Golgi trafficking, leading to Golgi fragmentation, ER swelling, and ER stress activation via ATF6. Functional assays revealed that GBF1 knockdown further decreased the expression of Golgi structural and trafficking-related proteins and exacerbated ER stress, whereas ATF6 silencing partially reversed these molecular alterations. Moreover, our results provide evidence that BA can stably bind to ATF6, inhibit its activation and proteolytic cleavage, restore the expression of Golgi and trafficking-related proteins, and reduce ER stress, thereby maintaining organelle homeostasis. In summary, our results identify the GBF1-ATF6 axis as a key pathway in Cu-induced ER-Golgi dysregulation and nephrotoxicity, and provide mechanistic insight into the protective role of BA in restoring subcellular homeostasis.

Graphical abstract

Introduction

Copper (Cu) is a persistent and essential trace metal that plays vital physiological roles in animals, but its widespread industrial and agricultural use has led to severe environmental contamination (Tapiero et al., 2003). Cu pollution has been listed as one of the 275 priority-controlled contaminants by the United States Environmental Protection Agency (USEPA), and is a matter of global concern. According to the International Copper Association, Cu consumption in 2022 was nearly 30% higher than in 2010, and global reserves reached approximately 1 billion metric tons by 2023 (Mejias et al., 2025). Recent surveys in the United States reveal alarming soil Cu concentrations in contaminated areas. For example, soils from industrial/urban gardens adjacent to smelters and industrial emissions demonstrated Cu levels of 59.4-388.8 mg/kg, substantially exceeding typical background levels (often < 50 mg/kg for U.S. soils) by more than 4 to 7 fold (Rajput et al., 2025). Similarly, surface water samples collected from the Pearl River Delta in China reported Cu concentrations up to 0.46 mg/L, a level exceeding the World Health Organization (WHO) guideline for drinking water (0.10 mg/L) by more than fourfold, raising concerns about aquatic ecosystem safety and human exposure risk (Fan et al., 2022). Although essential for animal health, chronic exposure to Cu through contaminated feed or water disrupts metal homeostasis and leads to systemic toxicity (Chen et al., 2022; Gao et al., 2024; Guo et al., 2022; Peng et al., 2024). Previous evidence has shown that, compared with other organs, the kidney is particularly susceptible to Cu accumulation (Zou et al., 2024). Despite clear evidence of Cu-induced glomerular atrophy, tubular dilation, epithelial degeneration, and impaired filtration in mammalian models, this aspect of nephrotoxicity remains insufficiently studied (Wang et al., 2022). Most previous studies have focused on histopathological changes and oxidative stress as primary indicators of Cu-induced renal damage. More recent findings have begun to explore mitochondrial dysfunction, autophagic disturbances, and apoptotic signaling in Cu-exposed renal cells, suggesting that subcellular organelle crosstalk may play a critical role in driving nephrotoxicity (Liu et al., 2021). Among these, the endoplasmic reticulum (ER) has emerged as a key target of metal-induced stress, due to its central role in protein folding and intracellular signaling. However, the integrity of ER-Golgi trafficking, which is essential for protein quality control and secretory pathway homeostasis, remains largely overlooked in the context of Cu-induced kidney injury. Disruption of ER-Golgi transport can lead to Golgi fragmentation, accumulation of misfolded proteins, and persistent ER stress—events that are increasingly linked to pathological outcomes in renal tissues. Despite growing evidence of ER stress involvement, how Cu perturbs ER-Golgi dynamics and whether this contributes to renal injury remain poorly understood.

Given this knowledge gap, attention has increasingly focused on the ER and Golgi, two central hubs of the eukaryotic secretory pathway that are functionally interconnected through vesicular transport mediated by COPI/COPII complexes and the ER–Golgi intermediate compartment (ERGIC). These organelles collectively regulate protein synthesis, modification, and trafficking, with newly synthesized proteins sequentially exported from the ER to the Golgi for further processing (Brandizzi and Barlowe, 2013; Watanabe et al., 2024). Beyond their canonical roles in secretion, accumulating evidence indicates that dynamic ER-Golgi interactions act as a critical regulatory node for cellular adaptation to stress (Dutta et al., 2024; Ragagnin et al., 2023; Zhao et al., 2018). Under ER stress, the PERK, IRE1, and ATF6 branches induce the expression of chaperones such as BiP/GRP78, often accompanied by Golgi fragmentation and abnormal glycosylation enzyme activity (Han et al., 2017). Prolonged pathological ER stress, as observed in neurodegenerative and metabolic disorders, is closely associated with Golgi depolymerization and dysfunction (Macke et al., 2024; Mendoza-Ferreira et al., 2020). Nevertheless, most studies have examined stress responses within individual organelles, and the molecular mechanisms underlying ER-Golgi crosstalk in toxicological contexts, particularly under heavy metal exposure, remain poorly defined.

Baicalin (BA), a flavonoid isolated from Scutellaria baicalensis, exhibits a wide spectrum of pharmacological activities, including anti-inflammatory, antioxidant, antiviral, and antitumor effects (Wang et al., 2024; Wen et al., 2023). Notably, BA has demonstrated protective actions in neurodegenerative, cardiovascular, renal, and metabolic disorders (Li et al., 2025; Zeng et al., 2024), yet the molecular mechanisms underlying its protective potential against heavy metal toxicity remain incompletely understood. In this context, targeting organelle crosstalk has emerged as a promising therapeutic strategy for mitigating heavy metal–induced toxicity, and natural products, with their structural diversity and broad bioactivities, represent a valuable resource for drug discovery. Critical questions therefore arise: Does Cu-induced nephrotoxicity involve disruption of ER-Golgi trafficking? Does BA counteract these pathological alterations by targeting specific signaling nodes? Can BA thereby restore organelle homeostasis and alleviate renal injury? Given the ecological and health concerns of Cu contamination, this study established in vivo duck kidney and in vitro primary renal tubular epithelial cell models of Cu exposure, combined with BA intervention, to address three objectives: elucidate the role of BA in Cu-induced disruption of ER-Golgi trafficking; determine how BA restores secretory pathway homeostasis under Cu-triggered ER stress; and evaluate its protective effects against Cu-mediated renal injury. Through an integrated strategy of molecular modeling, siRNA interference, and functional validation assays, we systematically investigated the interplay among ER-Golgi transport disorder, ER stress, and nephrotoxicity, while delineating the counteractive mechanisms of BA. Our findings reveal novel mechanistic insights into how BA stabilizes ER-Golgi dynamics and alleviates Cu-induced renal injury, supporting its potential as a therapeutic modulator of organelle crosstalk.

Materials and methods

Animals and treatment

This study was approved by the Animal Ethics Committee of Jiangxi Agricultural University (Approval No. JXAULL-2024-09-02). Prior to experimentation, all ducks were clinically examined to confirm their health status. A total of 48 one-day-old Peking ducks were randomly divided into four groups (n = 12 per group; equal numbers of males and females): (1) control group, (2) Cu group (400 mg Cu/kg diet, supplied as CuSO₄), (3) Cu+BA group (400 mg Cu/kg diet, supplied as CuSO₄, + 20 mg/kg baicalin), and (4) BA group (20 mg/kg baicalin). The amount of CuSO₄ added to the basal diet was calculated based on its Cu content to achieve a final Cu concentration of 400 mg/kg diet. The basal diet was formulated according to NRC (1994) guidelines. Dosages of Cu and BA were selected based on previous studies (Cheng et al., 2022; Wang et al., 2021). For the Cu+BA and BA groups, BA was thoroughly mixed into the basal diet and continuously provided throughout the 42-day experimental period. Throughout the experimental period, ducks had free access to their respective experimental diets and water.

Sample collection

On day 42, all ducks were fasted for 12 h before sample collection, with free access to water. Blood sampling and euthanasia were performed during the same time period to minimize potential variation associated with feeding status and circadian rhythms. Blood samples were collected using anticoagulant-free vacuum tubes for serum isolation. Ducks were subsequently euthanized with an overdose of pentobarbital sodium, and kidney tissues were harvested, rinsed in saline, and preserved appropriately. Samples were either stored at −80°C, fixed in 4% paraformaldehyde, or fixed in 2.5% glutaraldehyde.

Network toxicology

Network toxicology was used as a hypothesis-generating approach to identify biological processes potentially linking Cu exposure to kidney injury and to provide a basis for subsequent mechanistic investigation. The terms “Copper” and “kidney injury” were respectively queried in the CTD (https://ctdbase.org/) and GeneCards (https://www.genecards.org/), databases. The overlapping genes obtained from both datasets were identified as potential targets related to Cu-induced nephrotoxicity, followed by GO and KEGG enrichment analyses conducted using Genedenovo (https://www.omicshare.com/tools/).

Cu Distribution Map

To identify the global residue and production patterns of Cu, literature from 2023 to 2024 was systematically reviewed through the NCBI website (https://www.ncbi.nlm.nih.gov/) and the China National Knowledge Infrastructure (http://www.cnki.net) using the keywords “Cu”, “Environment”, and “Pollution”. Based on this literature screening, countries involved in Cu production were identified and categorized. In addition, 2023–2024 refined Cu production data obtained from Nasdaq (https://www.nasdaq.com/) were used to generate the graphical representation of the top ten Cu-producing countries.

Morphological observation

Kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and subjected to H&E staining for histopathological observation. Other samples were preserved in 2.5% glutaraldehyde, embedded in epoxy resin, and sectioned for ultrastructural examination using transmission electron microscopy (TEM) (Thermo Fisher, USA) (Xiong et al., 2025).

Serum biochemistry

Serum levels of creatinine, uric acid, and blood urea nitrogen (BUN) were measured using a Hitachi 3100 biochemical analyzer (Hitachi, Japan).

Antioxidant assays

Fresh kidney tissues were collected to determine total superoxide dismutase (T-SOD), catalase (CAT) and hydrogen peroxide (H₂O₂) levels according to the manufacturer’s instructions. The protein concentration of each kidney tissue homogenate was determined and adjusted to the same level before the T-SOD, CAT, and H₂O₂ assays. All assay kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Cell isolation and culture

Kidneys from 10 to 15-day-old ducks were excised, minced into small pieces, and digested with type I collagenase (5 mL) at 37°C for 8 min. The digested tissues were filtered through a 200-mesh sieve and centrifuged to obtain renal tubular epithelial cells. Cells were cultured in low-glucose DMEM (Solarbio,China) supplemented with 10% fetal bovine serum (FBS) (Bioind, Israel) at 37°C in a 5% CO₂ incubator. After 36 h, cells were divided into four groups: control, Cu (200 μM CuSO₄), Cu+BA (200 μM CuSO₄ + 25 μM BA), and BA (25 μM BA). Cells were treated for 12 h, and concentrations were selected based on previous study (Wang et al., 2022) and CCK-8 (Glpbio, USA) assay results.

siRNA intervention

Small interfering RNAs (siRNAs) targeting GBF1 and ATF6 were synthesized by Hanbio Biotechnology (Shanghai, China), and their detailed oligonucleotide sequences are listed in Table S1. Transfections were performed using RNAfit™ reagent (Hanbio, Shanghai, China) according to the manufacturer’s protocol. The final siRNA concentration was 50 nM, and subsequent treatment was initiated 6 h after transfection.

Immunofluorescence

Renal tubular epithelial cells grown on coverslips were fixed, permeabilized, and incubated overnight with primary antibodies against GM130 (Proteintech, China) and ATF6 (ABclonal, China), or SEC23 (Service Bio, China) and COPA (Proteintech, China). After PBS washing, cells were incubated with fluorescent secondary antibodies and counterstained with DAPI (Service Bio, China) for nuclear visualization.

Immunohistochemistry

Paraffin sections of kidney tissue were deparaffinized, rehydrated, and processed for antigen retrieval. Endogenous peroxidase activity was quenched, after which the sections were incubated with an anti-GRP78 primary antibody (Servicebio, China) and subsequently with an HRP-labeled secondary antibody. The immunoreactive signals were visualized using a DAB chromogenic substrate and observed under a microscope.

Molecular docking

Molecular docking analysis was conducted via CB-Dock, a platform based on AutoDock Vina, to predict the interaction between baicalin and ATF6 (Paggi et al., 2024). The three-dimensional structure of the receptor was retrieved from the Protein Data Bank (PDB), while the ligand structure was obtained from PubChem.

Molecular dynamics (MD) simulation

MD simulations were conducted using GROMACS 2022.3 (Van Der Spoel et al., 2005). Small molecules were parameterized with GAFF using AmberTools22, and RESP charges were calculated with Gaussian 16 W. The Amber99sb-ildn force field and TIP3P water model were applied. The system was neutralized with Na⁺ ions, minimized by steepest descent, and equilibrated under NVT and NPT ensembles. A 100 ns production run (2 fs step size) was performed. Trajectories were analyzed for RMSD, RMSF, radius of gyration, and free energy profiles (Kumar and Yaduvanshi, 2023).

Quantitative real‑time PCR (RT‑qPCR)

The primer sequences are provided in Supplementary Table S2. Total RNA was isolated from kidney tissues and subsequently reverse-transcribed into cDNA. Quantitative real-time PCR was carried out according to standard protocols, with β-actin serving as the internal control. Relative gene expression levels were determined using the 2^{^−ΔΔCt} method.

Western blotting

Kidney tissues were homogenized in RIPA buffer, and proteins were quantified using a BCA assay kit. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skimmed milk, membranes were incubated with primary antibodies against GM130 (1:5000; Proteintech, China), GRP78 (1:2000; Wanlei Bio, China), CHOP (1:2000; Wanlei Bio, China), GBF1 (1:1000; Proteintech, China), COPA (1:2000; Proteintech, China), ARF1 (1:2000; Uping Bio, China), ERGIC53 (1:2000; Uping Bio, China), SAR1 (1:2000; Uping Bio, China), ATF6 (1:2000; ABclonal, China) and GAPDH (1:50000; Proteintech, China) at 4°C overnight. After incubation with secondary antibodies, signals were visualized by ECL and quantified with ImageJ software.

Statistical analyses

Data were processed using Microsoft Excel 2021 and analyzed with GraphPad Prism (GraphPad Software, La Jolla, CA, USA). One-way ANOVA followed by Tukey’s multiple comparison test was used. Data are presented as mean ± SD of at least three independent experiments (n ≥ 3). Differences were considered significant at P < 0.05 (P < 0.05, P < 0.01, P < 0.001, two-tailed).

Results

Global distribution of Cu and network toxicology analysis reveal its environmental occurrence and potential nephrotoxic mechanisms

To provide environmental context for Cu exposure and to generate candidate hypotheses for its nephrotoxic mechanisms, we first summarized the global distribution of Cu production and recent pollution reports, followed by network toxicology analysis. A geographic distribution map was constructed based on multi-source data, illustrating the worldwide distribution of Cu-producing countries and regions (Fig. 1A). Among these, the top ten Cu-producing countries were identified using refined production data from 2023, with Chile, Peru, and China ranking highest (Fig. 1B). In parallel, a frequency map of Cu-related environmental reports from 2023 to 2024 was generated through literature and global media database screening, revealing hotspots of Cu residue events, particularly in industrial and agricultural regions (Fig. 1C). To further elucidate the potential toxicological mechanisms by which Cu contributes to kidney injury, network toxicology analysis was performed. A total of 1,417 Cu-related genes and 5,811 kidney injury-related genes were retrieved from the CTD and GeneCards databases. Venn diagram analysis identified 1,138 overlapping targets (Fig. 1D). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that these targets were mainly enriched in cancer pathways, AGE-RAGE signaling, TNF signaling, as well as pathways related to transport and catabolism (Fig. 1E, G, H). Notably, the enrichment of transport and catabolism pathways suggests a potential involvement of disrupted intracellular trafficking processes, possibly including ER-Golgi transport. Gene Ontology (GO) enrichment analysis further demonstrated that, in terms of biological processes (BP), the genes were primarily associated with biological regulation, cellular processes, response to stimuli, and metabolic processes. In the cellular component (CC) category, the genes were mainly localized to cellular anatomical entities and protein-containing complexes (Fig. 1F). Collectively, these results suggest that Cu may contribute to kidney injury by affecting biological regulation, stress responses, and metabolic processes of renal tubular epithelial cells, potentially through alterations in intracellular trafficking networks such as ER-Golgi transport, alongside pathways related to apoptosis, inflammation, and chemokine signaling.

Fig. 1.

Global distribution and network toxicology analysis of Cu. A) Cu-producing countries and regions worldwide in 2024. B) Top ten Cu-producing countries in 2024. C) Frequency map of Cu-related environmental reports worldwide from 2023 to 2024. D) Venn diagram for screening Cu-induced kidney damage related genes. E) PPI network diagram of Cu-induced kidney damage related genes. F) GO enrichment circle diagram of Cu-induced kidney damage related genes. G, H) KEGG enrichment results of Cu-induced kidney damage related genes.

BA attenuates Cu-induced kidney injury and oxidative stress in ducks

To investigate the nephrotoxicity induced by excessive Cu exposure and the protective effects of BA, we established a duck model of Cu exposure with BA intervention (Fig. 2A). Cu exposure significantly reduced both body and kidney weight in ducks, without noticeable changes in gross kidney morphology, which were reversed by BA treatment (Fig. 2B-D). Serum biochemistry analysis revealed that renal injury markers, including uric acid, urea nitrogen, and creatinine, were significantly elevated in the Cu-treated group but attenuated by BA intervention (Fig. 2E-G). In addition, Cu exposure markedly impaired renal antioxidant capacity, as evidenced by decreased T-SOD and CAT levels, together with elevated H₂O₂ content. BA supplementation effectively restored these parameters (Fig. 2H-J). Hematoxylin and eosin (H&E) staining demonstrated severe tubular dilatation and vacuolar degeneration of renal tubular epithelial cells in the Cu-treated group, which was alleviated by BA intervention (Fig. 2K). Ultrastructural analysis further revealed Golgi fragmentation with accumulation of vesicle-like structures, accompanied by ER swelling in the Cu-treated group, while these abnormalities were ameliorated by BA treatment (Fig. 2L).

Fig. 2.

BA attenuates Cu-induced kidney injury and oxidative stress in ducks. A) Schematic of Cu exposure model. B) Body weight of ducks. C) Kidney weight of ducks. D) Appearance of duck kidney (scale bar is 20 mm). E) Serum uric acid level. F) Serum urea level. G) Serum creatinine level. H-J) The activities of CAT and T-SOD, and the content of H₂O₂. K) Histopathology of the kidney. Tubular dilation (green arrow), vacuolar degeneration (red arrow). The scale bar is 100 μm. L) Ultrastructural observations on the ER and Golgi apparatus in the kidney. Swelling of the ER (yellow arrow), disruption of the Golgi apparatus (blue arrow). The scale bar is 200 nm. The data for each group were expressed as mean±SD (n ≥ 3). *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001 (compared with the control group); ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

BA alleviates Cu-induced Golgi fragmentation, ER stress, and ER–Golgi transport blockade in duck renal tubular epithelial cells

To further investigate the molecular mechanisms of Cu-induced renal injury and the protective effects of BA, we established an in vitro model using primary duck renal tubular epithelial cells (Fig. 3A). Immunofluorescence staining of GM130 revealed that Cu exposure caused pronounced Golgi fragmentation (Fig. 3B). Concurrently, co-localization immunofluorescence showed that the puncta and spatial distribution of SEC23 (COPII component) and COPA (COPI component) were significantly decreased, indicating impaired vesicular transport. These changes were partially reversed by BA treatment (Fig. 3C). Transmission electron microscopy further confirmed structural damage to both the Golgi apparatus and the ER following Cu exposure, whereas BA treatment alleviated these ultrastructural lesions (Fig. 3D). In addition, confocal microscopy demonstrated that Cu disrupted the spatial continuity between the ER and Golgi, while BA restored their close spatial proximity (Fig. 3S), supporting a structural basis for ER-Golgi communication disorder under Cu stress.

Fig. 3.

BA alleviates Cu-induced injury in duck renal tubular epithelial cells. A) Schematic diagram of renal tubular epithelial cell isolation process. B) Immunofluorescence of GM130 (scale bar is 10 μm). C) Immunofluorescence of SEC23 and COPA (scale bar is 20 μm). D) Ultrastructural observation of the ER and Golgi apparatus in renal tubular epithelial cells. Swelling of the endoplasmic reticulum (yellow arrow), disruption of the Golgi apparatus (blue arrow). The scale bar is 200 nm. E-R) Expression levels of proteins and mRNAs related to Golgi structure, ER stress, and ER-Golgi transport. S) Immunofluorescence observation of the ER and Golgi co-localization. The scale bar is 10 μm. *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001 (compared with the control group); ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

We next assessed the extent of ER stress. As expected, Cu exposure markedly upregulated the mRNA and protein levels of key ER stress markers GRP78 (encoded by HSPA5) and CHOP (encoded by DDIT3), whereas BA treatment significantly reduced their expression (Fig. 3F, M). To explore Golgi-associated protein dysregulation, we examined the expression of structural and functional components of the Golgi apparatus. Cu exposure markedly downregulated the mRNA levels of GORASP1 (trans-stacking protein), GOLGA2 (cis-stacking protein), and GBF1 (Golgi-specific BFA resistance factor 1), as well as the protein expression of GM130 (encoded by GOLGA2) and GBF1. BA treatment effectively alleviated these deficits (Fig. 3E, P-R). Given the essential role of vesicular trafficking in maintaining ER–Golgi dynamics, we analyzed key components of COPII (SAR1, SEC23, SEC13), COPI (ARF1, COPA), and ER-Golgi intermediate compartment (ERGIC; marker: ERGIC53/LMAN1). Cu exposure significantly downregulated the mRNA expression of SAR1, SEC13, SEC23, LMAN1, ARF1, and COPA, along with their corresponding protein levels. These changes were partially rescued by BA intervention (Fig. 3G-L, N, O). Together, these findings demonstrate that Cu disrupts Golgi structural integrity, induces ER stress, and impairs ER-Golgi vesicular transport in duck renal tubular epithelial cells. BA treatment alleviates these pathological alterations by restoring organelle structure, spatial proximity, and trafficking flux between the ER and Golgi.

Cu exposure and GBF1 knockdown disrupt Golgi and ER-Golgi transport and activate ATF6 in duck renal tubular epithelial cells

To elucidate how Cu disrupts Golgi and ER homeostasis, we first constructed a PPI network integrating Golgi structure, ER stress, and ER-Golgi transport–related factors, which identified GBF1 as a central node (Fig. 4A). Ultrastructural analysis confirmed that combined Cu exposure and GBF1 knockdown (siGBF1) resulted in severe Golgi fragmentation and ER swelling, consistent with aggravated organelle dysfunction (Fig. 4B). Confocal immunofluorescence further supported these observations: the fluorescence intensity of GM130 was diminished and dispersed, while ATF6 signals were markedly enhanced following siGBF1 and Cu co-treatment (Fig. 4L). In addition, co-localization imaging showed that ER-Golgi spatial association was disrupted under these conditions (Fig. 4C). At the molecular level, siGBF1 reduced the expression of Golgi structural proteins and ER-Golgi transport–related factors, while upregulating ER stress markers, with Cu co-treatment further intensifying these alterations (Fig. 4D-J). Notably, ATF6, a major ER stress sensor, was strongly activated by GBF1 knockdown, suggesting that Golgi dysfunction directly contributes to ATF6 signaling. Within the ER-Golgi transport machinery, siGBF1 predominantly downregulated COPI-associated components (ARF1, COPA), whereas COPII factors (SAR1, SEC23, SEC13) and the ERGIC marker LMAN1/ERGIC53 were less affected. Consistent with this, punctate signals of SEC23 and COPA were markedly reduced in the siGBF1 and Cu groups, indicating impaired vesicular transport (Fig. 4K). Collectively, these results demonstrate that GBF1 is a key regulator of Golgi structural maintenance and ER-Golgi trafficking. Its suppression exacerbates Cu-induced Golgi fragmentation, ER stress, and impaired organelle communication, in part through activation of the ATF6 signaling pathway.

Fig. 4.

GBF1 knockdown exacerbates Cu-induced disruption of ER-Golgi transport in duck renal tubular epithelial cells. A) PPI network diagram of Golgi apparatus structure, ER stress, and ER-Golgi transport-related factors. B) Ultrastructural observation of the ER and Golgi apparatus in renal tubular epithelial cells. Swelling of the endoplasmic reticulum (yellow arrow), disruption of the Golgi apparatus (blue arrow). The scale bar is 200 nm. C) Immunofluorescence observation of the ER and Golgi co-localization. The scale bar is 10 μm. d-J) Expression levels of proteins and mRNAs related to Golgi structure, ER stress, and ER-Golgi transport. K) Immunofluorescence of SEC23 and COPA (scale bar is 20 μm). L) Immunofluorescence of GM130 and ATF6 (scale bar is 20 μm). *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001 (compared with the control group); ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

ATF6 knockdown alleviates Cu-induced ER stress and Golgi fragmentation and restores ER-Golgi transport in duck renal tubular epithelial cells

To clarify the role of ATF6 in Cu-induced cellular injury, we established a Cu exposure model combined with ATF6 knockdown (siATF6). Transmission electron microscopy demonstrated that siATF6 attenuated Cu-induced ultrastructural abnormalities, including ER swelling and Golgi fragmentation (Fig. 5A). Consistently, molecular analyses revealed that siATF6 markedly reduced the expression of ER stress markers while partially restoring the expression of Golgi structural proteins and ER–Golgi transport–related factors at both the mRNA and protein levels (Fig. 5B–K). Immunofluorescence confirmed that ATF6 fluorescence intensity was diminished in the siATF6 group, accompanied by preservation of a more compact Golgi morphology (Fig. 5L). In addition, the increased fluorescence puncta of transport-related proteins indicated that ER-Golgi transport flux, which had been impaired by Cu exposure, was partially restored by ATF6 knockdown (Fig. 5M). Furthermore, ER-Golgi co-staining revealed a clearer spatial continuity between the two organelles in siATF6-treated cells compared with the Cu group (Fig. 5N). These results highlight a central role of ATF6 in mediating Cu-induced ER-Golgi dysfunction and demonstrate that its inhibition can restore organelle integrity and transport balance.

Fig. 5.

Knockdown of ATF6 improves ER- Golgi transport in duck renal tubular epithelial cells. A) Ultrastructural observation of the ER and Golgi apparatus in renal tubular epithelial cells. Swelling of the endoplasmic reticulum (yellow arrow), disruption of the Golgi apparatus (blue arrow). The scale bar is 200 nm. B-K) Expression levels of proteins and mRNAs related to Golgi structure, ER stress, and ER-Golgi transport. L) Immunofluorescence of GM130 and ATF6 (scale bar is 20 μm). M) Immunofluorescence of SEC23 and COPA (scale bar is 20 μm). N) Immunofluorescence observation of the ER and Golgi co-localization. The scale bar is 10 μm. *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001 (compared with the control group); ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

BA restores ER-Golgi transport disrupted by Cu exposure via targeting ATF6 in duck renal tubular epithelial cells

To investigate the mechanism by which BA mitigates Cu-induced ER-Golgi transport impairment, molecular docking and dynamics simulations were performed, revealing a strong and stable interaction between BA and ATF6 (Fig. 6A-F). In vitro, BA treatment under Cu exposure alleviated the abnormal upregulation of ATF6 at both the mRNA and protein levels (Fig. 6G, I, J). Immunofluorescence analysis further confirmed that BA reduced ATF6 fluorescence intensity and preserved a more compact Golgi morphology, while ER-Golgi co-staining demonstrated improved spatial continuity between the two organelles (Fig. 6H). Subsequently, in vivo validation was performed, and the results were consistent with the in vitro findings. Immunohistochemistry results showed that Cu exposure increased GRP78-positive areas in duck kidney tissue (Fig. 6L). Additionally, Cu exposure also upregulated the expression levels of ER stress-related factors, while reducing the expression levels of Golgi structural and ER-Golgi transport-related factors; these changes were partially reversed by BA treatment (Fig. 6K, M-R). These results suggest that BA may interact with ATF6 to inhibit its activation, thereby alleviating ER stress and restoring ER-Golgi transport under Cu exposure.

Fig. 6.

BA restores Cu disrupted ER-Golgi transport by targeting ATF6 in duck renal tubular epithelial cells. A) BA directly bound to ATF6. B-F) MD simulations verifying the molecular docking results between ATF6 and BA. G, I-J) Protein and mRNA expression levels of ATF6, ATF6-p90, and ATF6-p50. H) Immunofluorescence of GM130 and ATF6 (scale bar is 20 μm). K) Expression levels of mRNAs related to Golgi structure, ER stress, and ER-Golgi transport. L) GRP78 immunohistochemistry (scale bar is 100 μm). M-R) Expression levels of proteins related to Golgi structure, ER stress, and ER-Golgi transport. *P < 0.05, ^⁎⁎P < 0.01, ^⁎⁎⁎P < 0.001 (compared with the control group); ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Discussion

Although increasing attention has been paid to Cu pollution and its adverse effects on aquatic and terrestrial animals, our understanding of organelle-level mechanisms underlying Cu-induced nephrotoxicity remains insufficient (Dong et al., 2024; Shang et al., 2025; Zhang et al., 2025a; Zheng et al., 2023). With the advancement of research, the metabolic toxicity of Cu is being increasingly recognized; however, the current focus remains disproportionately centered on oxidative stress and individual cell damage, with insufficient attention paid to ER-Golgi crosstalk and kidney-specific injury mechanisms (Dong et al., 2025; Lian et al., 2023; Liu et al., 2024; Wang et al., 2025). Given that the kidney plays a central role in Cu excretion and is particularly vulnerable to metal-induced injury, this study systematically investigated Cu-triggered renal damage and interactions between associated organelles in ducks. Early studies have demonstrated that the cytosolic Cu chaperone ATOX1 transports Cu to the Golgi/trans-Golgi network (TGN), where Cu-transporting ATPases ATP7A and ATP7B facilitate the incorporation of Cu into cuproenzymes (Chen et al., 2025; Masaldan et al., 2018; Wang and Weinman, 2004). ATP7A and ATP7B export excess Cu via vesicular trafficking to maintain homeostasis when cellular Cu levels exceed normal thresholds (Gerbasi et al., 2003). Although previous studies have highlighted the importance of the Cu–Golgi axis in maintaining intracellular metal trafficking, little is known about how Cu exposure affects ER-Golgi communication under pathological conditions (Hua et al., 2025; Yuan et al., 2022). In our study, Cu exposure led to severe renal impairment, characterized by significant weight loss, elevated renal injury markers, and histopathological alterations such as tubular dilation, epithelial vacuolization, Golgi fragmentation, and ER swelling. These morphological hallmarks suggest that Cu-induced nephrotoxicity may involve trafficking disruption and protein-folding stress, potentially mediated by ER-Golgi crosstalk dysfunction.

Our primary findings from network toxicology analysis revealed that Cu exposure predominantly affects biological processes associated with organelle function, particularly intracellular transport and catabolic metabolism, and subsequent experiments confirmed significant structural alterations in both the Golgi apparatus and endoplasmic reticulum, leading us to hypothesize that ER-Golgi trafficking disruption is a central contributor to Cu-induced renal injury. Several lines of evidence support the role of secretory pathway dysfunction in Cu toxicity. For example, Mutations in Cu-transporting ATPases (ATP7A and ATP7B) cause systemic Cu misdistribution and severe organ damage, as seen in Menkes and Wilson diseases (Delafontaine et al., 2024; Shetty and Upadhya, 2016). Recent studies further indicate that Cu exposure causes Golgi collapse and secretion defects in hepatic and neuronal models (Petruzzelli et al., 2022; Scheiber et al., 2014; Tian et al., 2025). Consistent with these reports, we observed that Cu treatment induced prominent Golgi fragmentation and ER swelling in duck renal tubular epithelial cells, accompanied by upregulation of ER stress markers GRP78 and CHOP. Cu exposure also significantly downregulated the expression of key ER–Golgi transport components, including ARF1 and COPA (COPI), SAR1 (COPII), and ERGIC53 (ERGIC), at both transcript and protein levels. Reduced SEC23-COPA co-localization further indicated trafficking blockade. These results collectively suggest that Cu disrupts the ER-Golgi transport machinery at multiple levels, intensifying protein-folding stress and ER dysfunction. Mechanistically, we identified GBF1 as a central upstream regulator in this cascade. Knockdown of GBF1 phenocopied Cu-induced cellular alterations, including Golgi disassembly, trafficking impairment, and ATF6 activation, supporting the notion that GBF1 downregulation is an initiating event in ER-Golgi disruption under Cu stress. These observations delineate a mechanistic pathway in which Cu suppresses GBF1 expression, destabilizing COPI-mediated retrograde transport and triggering sustained ER stress responses. Notably, prolonged ER stress driven by the GBF1-ATF6 axis has also been associated with inflammatory signaling and apoptosis in renal tubular epithelial cells (Yu et al., 2024), suggesting that the consequences of Cu-induced ER-Golgi dysfunction extend beyond proteostasis imbalance to downstream pathological injury.

Building on this established mechanism, we next turned our focus to downstream stress transducers, particularly ATF6, a key branch of the unfolded protein response (UPR) that is highly dependent on ER-Golgi trafficking (Parchure et al., 2025). Under normal conditions, ATF6 is transported to the Golgi and sequentially cleaved by S1P and S2P proteases to release its active p50 fragment, which then enters the nucleus to initiate adaptive gene expression (Farhan et al., 2025; Senft and Ronai, 2015). While trafficking inhibition typically blocks this process, we observed paradoxical ATF6 activation despite ER-Golgi blockade, suggesting an alternative activation mechanism. One plausible explanation is that Cu-induced Golgi fragmentation causes mislocalization of S1P/S2P into the ER, allowing noncanonical ATF6 cleavage, as previously reported under BFA treatment and in certain malignancies (B R et al., 2023; Freemantle et al., 2024). Although this hypothesis requires further validation, our data strongly support trafficking-independent ATF6 activation. Functionally, ATF6 knockdown significantly reversed Cu-induced ER stress, restored COPI/COPII gene expression, and reduced Golgi fragmentation, indicating that ATF6, while protective under transient stress, may shift toward a pro-apoptotic role under persistent Cu exposure (Papaioannou et al., 2018). In line with this, sustained ATF6-CHOP signaling has been reported to promote pro-inflammatory cytokine release and apoptotic pathways in stressed renal tubular epithelial and hepatic cells (Brenner et al., 2013; Sun et al., 2025), including increased secretion of IL-1β and TNF-α, which may represent critical mediators linking ER-Golgi dysfunction to renal tissue injury.

Given the dual role of ATF6 in cellular fate decisions, we next explored whether pharmacological modulation of this pathway could confer therapeutic benefit. Naturally derived compounds, such as BA, have gained attention for their multi-target potential and low toxicity in heavy metal toxicity models (Coleman et al., 2025; Esteve-Garcia et al., 2025). BA, a flavonoid glycoside from Scutellaria baicalensis, has previously shown protective effects via Cu chelation and ROS suppression (Fan et al., 2024; Priyono et al., 2024; Zhang et al., 2025b); however, its role in modulating organelle signaling remains largely unexplored. In our study, BA treatment alleviated Cu-induced renal injury both in vivo and in vitro, improving serum biochemical markers, reducing oxidative stress, and ameliorating ER and Golgi ultrastructural abnormalities. Molecular docking and dynamics simulations predicted a strong, stable interaction between BA and ATF6, suggesting possible direct engagement. Supporting this, BA treatment suppressed ATF6 and CHOP expression, restored COPI/COPII levels, and improved SEC23–COPA spatial co-localization, indicating enhanced ER-Golgi trafficking. These results imply that BA interrupts the self-amplifying cycle in which Cu-induced ER stress drives Golgi disintegration and further activates ATF6, thereby promoting apoptosis. Although further biophysical validation (e.g., surface plasmon resonance or nuclear tracking of ATF6-p50) is warranted, our findings, together with prior reports on BA-mediated UPR modulation, support the plausibility of this mechanism. Importantly, these insights not only elucidate how BA mitigates Cu-induced ER–Golgi dysfunction at the cellular level but also highlight ATF6 as a promising therapeutic target. This provides a mechanistic rationale for developing ATF6-targeted strategies to combat heavy metal–induced renal injury, with broader implications for public health, environmental toxicology, and food safety.

Conclusion

In summary, this study provides novel insights into ER–Golgi trafficking dysfunction as a central mechanism of Cu-induced nephrotoxicity. We demonstrated that Cu exposure suppresses GBF1 expression, thereby destabilizing the COPI complex and impairing ER-Golgi transport, which in turn drives Golgi fragmentation, exacerbates ER stress, and activates ATF6-CHOP signaling to promote renal tubular epithelial injury. Notably, BA mitigates these pathological alterations by targeting ATF6, attenuating ATF6 activation, reducing CHOP-mediated stress responses, and partially restoring ER-Golgi trafficking homeostasis. Collectively, our findings establish that BA alleviates Cu-induced renal damage in ducks through modulation of the GBF1-Golgi-ER-ATF6 axis, providing a mechanistic basis for ATF6-targeted interventions against heavy metal-induced nephrotoxicity.

Ethics declaration

This study was approved by the Animal Ethics Committee of Jiangxi Agricultural University (Approval No. JXAULL-2024-09-02).

Author Contributions

Hongyu Shang and Wei Tang conceived and designed the study, interpreted the findings, and drafted and revised the manuscript. Xueyan Dai and Huabin Cao participated in data collection and analysis, contributed to the interpretation of the results, and critically revised the manuscript for important intellectual content. Xiaona Gao and Chenghong Xing contributed to data collection, organization, and presentation of the findings, and critically reviewed and refined the manuscript. Guoliang Hu contributed to data acquisition, interpretation of the results, and provision of research resources, and critically reviewed the manuscript for important intellectual content. Fan Yang supervised and coordinated the study, secured funding and research resources, contributed to interpretation of the findings, and critically revised the manuscript. All authors reviewed and explicitly approved the final version submitted for publication. They are accountable for all aspects of the work and will ensure that any questions regarding the accuracy or integrity of any part of the article are appropriately investigated and resolved.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.