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. 2025 Dec 2;4(4):668–683. doi: 10.1021/envhealth.5c00494

The Role of Aryl Hydrocarbon Receptor-Dependent Signaling Pathway in the Profibrotic Effects of Dioxin-like Compounds in Human Renal Tubular Epithelial Cells

Ruihong Zhu †,‡, Yiyun Liu §, Xiaoxu Hu †,‡, Yongchao Ma †, Wei Yan †,‡, Li Xu †,‡, Heidi Qunhui Xie †,‡,*, Bin Zhao ‡, Junfa Yin †,‡, Hailin Wang †,‡
PMCID: PMC13097154  PMID: 42022190

Abstract

The aryl hydrocarbon receptor (AhR) signaling pathway mediates nephrotoxic compound effects on the kidney, although its mechanisms are incompletely understood. Given that renal tubulointerstitial fibrosis is a central pathological feature of progressive kidney diseases, we investigated AhR-induced profibrotic events at the molecular and cellular levels in human renal tubular epithelial cells (HKC). We found that the AhR activation by the potent agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) promoted epithelial–mesenchymal transition (EMT) and inflammatory responses. This was evidenced by a 56.19% decrease in E-cadherin; 1.87-, 2.39-, and 8.27-fold increases in fibronectin, MMP9, and IL-6, respectively; and a 37.77% enhancement in cell migration. Transcriptome analysis and experimental validations confirmed the consistent dysregulation of these markers. Moreover, we found the profibrotic effects of the known nephrotoxic phytochemical aristolactam I (AL-I) also involving activation of AhR and consistent regulation of the above marker genes, primarily via the AhR. In addition, the transcriptome data further suggested that AhR activation may indirectly induce the profibrotic epidermal growth factor receptor (EGFR) pathway by upregulating AREG, EREG, and TGF-α, indicating crosstalk between AhR and EGFR. Given the wide variety of AhR-active chemicals, these AhR-EMT/inflammation-related markers could be used to screen nephrotoxicity of emerging dioxin-like pollutants and toxic phytochemicals.

Keywords: aryl hydrocarbon receptor (AhR); renal fibrosis; epithelial−mesenchymal transition (EMT); aristolactam I (AL-I); 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

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1. Introduction

The aryl hydrocarbon receptor (AhR) is a highly evolutionarily conserved ligand-activated transcription factor that plays an important role in mediating the effects of various environmental pollutants and natural compounds. Notably, recent studies revealed that excessive AhR activation induced by the exogenous toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in neonatal kidneys can lead to severe hydronephrosis in C57BL/6J mice. Furthermore, it has been demonstrated that patients with chronic kidney disease (CKD) exhibit significantly higher serum AhR-activating potential (AhR-AP) than healthy controls. Tryptophan metabolites and aryl-containing metabolites, such as indole-3-acetic acid (IAA) and 1-aminopyrene, which accumulate in CKD patients, can bind to the AhR and regulate the transcription of both AhR and downstream genes, thereby promoting inflammation, glomerular injury, and renal fibrosis. , These findings suggest that the expression of AhR and the activity of AhR signaling are potentially associated with kidney diseases, including renal fibrosis.

Renal fibrosis, particularly tubulointerstitial fibrosis, is a common end point in almost all progressive CKD. This process can be achieved by converting renal tubular epithelial cells into fibroblasts through the epithelial–mesenchymal transition (EMT) and promoting the accumulation of extracellular matrix (ECM). Consequently, EMT plays a crucial role in the development of renal fibrosis. The key events of EMT include the loss of epithelial cell adhesion (e.g., downregulation of E-cadherin and zonula occludens-1 expression), the acquisition of mesenchymal cell characteristics (e.g., upregulation of fibronectin and alpha-smooth muscle (α-SMA) expression), reorganization of the cytoskeletal architecture with enhanced cell migration, and disruption of the tubular basement membrane by matrix metalloproteinases (MMPs). , Notably, the enhanced cell migration serves as a key morphological indicator for assessing the progression of EMT. These indicators have been widely recognized as hallmarks of renal fibrosis in numerous in vivo and in vitro studies. , The role of AhR in pathological and pathophysiological mechanisms for fibrosis has been investigated in diverse organs, such as liver, heart, and lungs. On the one hand, the function of endogenous activation of AhR in counteracting fibrosis has been reinforced, but on the other hand, it has been demonstrated that the AhR pathway mediates the dysregulation of EMT marker genes in response to EMT-promoting chemical(s). However, its functions and potential mechanisms in renal fibrosis remain unexplored, especially fibrosis or EMT induced by exogenous environmental pollutants and natural compounds. Pyo et al. and Hsieh et al. demonstrated that both the nephrotoxicant ochratoxin A (OTA) and the AhR ligand IS mediate renal EMT via the AhR pathway. ,

In addition, some AhR ligands have also been found to bypass AhR and exert toxic effects, and a large number of studies have reported that renal fibrosis is related to various signaling pathways, such as the EGFR, MAPK, and PI3K/Akt signaling pathways. − Therefore, paying attention to both AhR-direct and -indirect pathways in renal fibrosis research will open broader research prospects for disease mechanism analysis and treatment strategy development.

The epidermal growth factor receptor (EGFR/ErbB1), a transmembrane protein belonging to the tyrosine kinase receptor ErbB family, is expressed in various organs, including the kidney. It can be activated by various ligands, including epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin (AREG), and epiregulin (EREG). Most of these ligands have been identified in the kidneys and mediate complex physiological and pathological processes, such as kidney development, recovery from acute kidney injury (AKI), and mediation of renal injury. − Substantial evidence indicates that EGFR and its ligands play critical roles in renal fibrosis, particularly in renal EMT, through crosstalk with multiple signaling pathways, including TGF-β1/Smad, NF-κB, and ERK1/2. − Notably, AhR activation has been shown to drive EGFR signaling through two primary mechanisms in diverse cellular models, including human breast cancer cells, keratinocytes, head and neck squamous cell carcinoma (HNSCC) cells, and lung cancer cells: (I) AhR ligand (e.g., TCDD)-driven dissociation of the AhR complex releases the proto-oncogene tyrosine-protein kinase Src (c-Src), which can directly phosphorylate and activate EGFR or indirectly activate EGFR via sequential activation of protein kinase C (PKC) and sheddases, leading to ectodomain shedding of cell-surface-bound EGFR ligands; and (II) the canonical AhR pathway transcriptionally upregulates the expression of EGFR ligands (e.g., AREG and EREG), increasing the production of EGFR ligands. However, there is currently a lack of research on the crosstalk between AhR and EGFR in kidneys, with only Choi et al. reporting negative results for the expression of AREG and EREG in mouse fetal kidneys after in utero TCDD exposure.

Currently, the toxicological and pathological roles of AhR in renal fibrosis and EMT have been applied in the development of targeted therapeutic interventions. For example, geniposidic acid has been shown to alleviate renal fibrosis in rats with chronic tubulointerstitial nephropathy by inhibiting AhR nuclear translocation. Meanwhile, the nephrotoxic mechanisms of IS, an endogenous ligand of AhR, have partially indicated the potential nephrotoxic mechanisms of exogenous pollutants such as TCDD. Thus, we propose extending AhR-focused mechanistic research to certain naturally occurring nephrotoxins such as aristolochic acid analogues (AAAs). AAAs are a class of naturally occurring polyaromatic nitrogen compounds and abundant in the plants of the genera Aristolochia and Asarum, which are spread all over the world. For centuries, plants containing AAAs have been used as herbal medicine to treat various symptoms and diseases. Until the early 1990s, a group of female patients in Belgium developed rapidly progressive interstitial nephritis accompanied by urothelial carcinoma (UTUC) after ingesting slimming pills containing AAAs. This incident drew worldwide attention to the high nephrotoxicity and carcinogenicity of AAAs. Subsequently, similar cases were reported in some Eastern European countries on the Balkan Peninsula, which are also the result of exposure to AAAs. Recently, these diseases were described as CKD linked to environmental factors, and AAAs were regarded as emerging environmental pollutants, posing a risk of cocontaminating the environment with pollutants such as polycyclic aromatic hydrocarbons (PAHs) and affecting human health. Since both AAAs and persistent organic pollutants (POPs) can induce renal fibrosis, , and considering the involvement of AhR in fibrosis, we indicate that AhR may serve as a shared molecular target to alleviate nephrotoxicity during coexposure. To explore the role of AhR in renal fibrosis, we used renal tubular epithelial HKC cells as an in vitro model to investigate the effects of two typical environmental toxins, the classical AhR ligand TCDD and the AAA component aristolactam I (AL-I), in the process of renal fibrosis. Transcriptome analysis elucidated AhR-direct and -indirect regulatory mechanisms of renal fibrosis, particularly in EMT, and revealed crosstalk between the AhR and EGFR signaling pathways. Furthermore, this study extends the role of AhR from TCDD to emerging natural pollutants AAAs, providing new insight for investigating the mechanisms underlying AAA-induced renal fibrosis.

2. Materials and Methods

2.1. Reagents

The reagents used in this study were 2,3,7,8-TCDD (Wellington Laboratory, Ontario, Canada), dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St Louis, MO), and CH223191 (Sigma-Aldrich). The AAAs employed in this study included aristolochic acid A (AA-A), aristolochic acid B (AA-B), aristolochic acid C (AA-C), aristolochic acid D (AA-D), 7-hydroxyaristolochic acid A (AA-VIIA), aristololactam I (AL-I), and Aristolactam BII (AL-BII) all obtained from DeSiTe (Chengdu, China).

2.2. Cell Culture

Human renal tubular epithelial HKC cells were purchased from the cell resource center of the Chinese Academy of Medical Sciences (Beijing, China) and cultured in Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F-12) (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Corning, NY, USA) and 1% penicillin–streptomycin (P/S) (Gibco). The CBG2.8D cell line was purchased from Hangzhou Toxsense (Hangzhou, China) and cultured in α-minimum essential medium (α-MEM) (Gibco) supplemented with 10% FBS and 1% P/S. All cell lines were incubated at 37 °C in a normal humidified 5% CO2 incubator.

2.3. Cell Viability Assay

Cell viability was quantitatively assessed using a Cell Counting Kit-8 (CCK-8) (Sangon, Shanghai, China) according to the instruction manual. Briefly, HKC cells were seeded in 96-well plates at 1 × 104 cells/well in 100 μL of complete medium (10% FBS) and allowed to adhere for 24 h. Experimental groups included (1) TCDD (10–10–10–7 mol/L), (2) AAAs (10–10–10–5 mol/L), and (3) solvent control (0.1% DMSO), with six replicates per condition. After 24 or 48 h compound treatment, 10 μL of CCK-8 reagent was added directly to the culture medium in each well followed by incubation for 2 h under standard conditions (37 °C, 5% CO2, dark environment). Absorbance (450 nm) was recorded using a spectrometer (Tecan Infinite F200 Pro, Männedorf, Switzerland).

2.4. Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from HKC cells using the GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. RNA concentration and purity were determined spectrophotometrically via NanoDrop (2000/2000c, Thermo Fisher Scientific). The A 260/A 280 ratio was maintained between 1.8 and 2.2. Synthesis of cDNA was performed with 2 μg of total RNA using a RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Quantitative PCR was carried out in triplicate using GoTaq qPCR Master Mix (Promega, Madison, WI) on a QuantStudio 6 Flex Real-Time System (Thermo Fisher Scientific) with the following program: 95 °C for 120 s, 40 cycles of denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s, and extension at 72 °C for 20 s. Gene expression levels were normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) and quantified using the comparative threshold cycle (2–ΔΔCT) method. Primers were designed with the NCBI Primer-BLAST tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) using genomic sequences from GenBank (http://www.ncbi.nlm.nih.gov/genbank/). The primer synthesis was performed by Sangon. All primers exhibited 95–105% amplification efficiency, as validated by standard curve analyses. The primer sequences are shown in Table .

1. Primers Used for qPCR in This Study.

gene siRNA sequence
E-cadherin (NM_001317185.2) (F) 5′-ATTTTTCCCTCGACACCCGAT-3′
  (R) 5′-TCCCAGGCGTAGACCAAGA-3′
fibronectin (NM_001365523.2) (F) 5′-GGCTTGAACCAACCTACGGATGAC-3′
  (R) 5′-TCCTTCTGCCACTGTTCTCCTACG-3′
MMP-9 (NM_004994.3) (F) 5′-AGACCTGGGCAGATTCCAAAC-3′
  (R) 5′-CGGCAAGTCTTCCGAGTAGT-3′
IL-6 (NM_001318095.2) (F) 5′-TCCTTCTCCACAAACATGTAACAA-3′
  (R) 5′-CCATCTTTGGAAGGTTCAGGTTG-3′
AhR (NM_001621.5) (F) 5′-CAACAGCAACAGTCCTTGGC-3′
  (R) 5′-GTTGCTGTGGCTCCACTACT-3′
AhRR (NM_001377239.1) (F) 5′-AGGTTTGGTTGGCAGGACT-3′
  (R) 5′-GCTCAGATGGTTGGCTGTTC-3′
CYP1A1 (NM_001319216.2) (F) 5′-TCGGCCACGGAGTTTCTTC-3′
  (R) 5′-GGTCAGCATGTGCCCAATCA-3′
CYP1B1 (NM_000104.4) (F) 5′-CTCTGCTGGTCAGGTCCTTG-3′
  (R) 5′-CACTGCCAACACCTCTGTCT-3′
AREG (NM_001657.4) (F) 5′-GAGCCGACTATGACTACTCAGA-3′
  (R) 5′-TCACTTTCCGTCTTGTTTTGGG-3′
EREG (NM_001432.3) (F) 5′-GTGATTCCATCATGTATCCCAGG-3′
  (R) 5′-GCCATTCATGTCAGAGCTACACT-3′
TGFA (NM_001099691.3) (F) 5′-AGGTCCGAAAACACTGTGAGT-3′
  (R) 5′-AGCAAGCGGTTCTTCCCTTC-3′
FBN1 (NM_001406716.1) (F) 5′-GCGGAAATCAGTGTATTGTCCC-3′
  (R) 5′-CAGTGTTGTATGGATCTGGAGC-3′
CCL20 (NM_001130046.2) (F) 5′-GGCGAATCAGAAGCAAGCAA-3′
  (R) 5′-GATTTGCGCACACAGACAACT-3′
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2.5. Wound Healing Assay

Cell migration capacity was assessed through a wound healing assay. The protocol was mainly adopted from Chen et al. with minor modifications. The six-well plates were premarked with three parallel reference lines before cell seeding. Upon reaching 90–95% cell confluence, uniform linear wounds were made in the cell monolayers using 1000 μL pipet tips (Axygen, Union City, CA). Wound edges adjacent to the reference marks were selected as imaging regions. Following three gentle washes with phosphate-buffered saline (PBS) to remove disassociated cells and debris, we administered experimental treatments were administered. Phase-contrast images were captured at 0, 24, and 48 h post-treatment using an inverted microscope (Olympus, Tokyo, Japan) equipped with a digital SLR camera (Canon, Tokyo, Japan). Six nonoverlapping fields per well were captured along the parallel reference lines. Quantitative analysis was performed using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD).

2.6. Transcriptome Sequencing (RNA-Seq) Analysis

The total RNA of the treated HKC cells was extracted by a GeneJET RNA Purification Kit (Thermo Fisher Scientific) according to the instruction manual. After verification of integrity and purity, total RNA was randomly fractured into fragments of approximately 200 bp and transcribed to double-stranded DNA with added adaptors. The sequences of the resulting DNA products were probed through Illumina NovaSeq 6,000 by Majorbio (Beijing, China). The expression level of each transcript was calculated according to the transcript per million read (TPM) method before the identification of differentially expressed genes (DEGs) between each pair of samples. The DEG analysis was performed using DESeq2/DEGseq/EdgeR with Q ≤ 0.05, and DEGs with |log2 FC| > 1 and Q ≤ 0.05 (DESeq2 or EdgeR) or Q ≤ 0.001 (DEGseq) were considered to be significantly differentially expressed genes. − Gene Ontology (GO) functional enrichment analysis was performed by Fisher’s exact test using GOatools Software, and the calibrated p values were obtained by four methods, including Bonferroni, Holm, Sidak, and false discovery rate, to control the false positive rate. When the calibrated p value (p_fdr) was ≤0.05, these entries were considered statistically significant GO enrichment entries. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed by Fisher’s exact test using KOBAS software. Entries with calibrated p-values of ≤0.05 were considered statistically significant KEGG enrichment entries. Majorbio bioinformatic analysis cloud computing platform 4 was used to search and analyze the expression changes of genes. Enrichment circle plots and DEG chord diagrams were plotted by https://www.bioinformatics.com.cn, an online platform for data analysis and visualization. Enriched item chord diagrams were plotted by PlotDB.

2.7. AhR Activation Assay

The AhR-mediated activities of seven AAAs were tested using the CBG2.8D cell line and luciferase reporter gene assay. Cells were seeded in 96-well white-bottom clean plates at 2 × 104 cells/mL (100 μL/well) and allowed to adhere overnight. Experimental groups included the following: (1) background control: cell-free wells containing 100 μL of low-serum α-MEM (2% FBS); (2) solvent control: cell-containing wells with 100 μL of α-MEM containing 1% DMSO; (3) treatment groups: cell-containing wells with serially diluted compounds (10–10–10–5 mol/L in low-serum α-MEM). Following 24 h incubation, luciferase activity was quantified using the Luciferase Assay System (Promega, Madison, WI) according to the manufacturer’s protocol, and relative light units (RLUs) were immediately measured using a microplate reader (GloMax Multi+Detection System, Promega). A standard curve was generated using 2,3,7,8-TCDD (1.52 × 10–13 to 1 × 10–9 mol/L) on every plate for assay calibration and positive control validation.

2.8. Knockdown of AhR Gene Using Small Interfering RNA (siRNA)

The HKC cells were transfected with human AhR siRNA or nontargeting control siRNA (NC) before chemical treatment. The siRNAs were designed and synthesized by the SyngenTech Company (Beijing, China). Transfection was conducted using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. After transfection for 24 h, the medium containing siRNA and transfection reagent was replaced with fresh culture medium containing 10–6 mol/L AL-I. The final concentration of AhR siRNA or NC was 20 nmol/L. The sequences of siRNA used in this study are shown in Table .

2. siRNA Sequences Used in This Study.

gene siRNA sequence
NC 5′-UUC UCC GAA CGU GUC ACG UTT-3′
5′-ACG UGA CAC GUU CGG AGA ATT-3′
AhR 5′-GAA CUU ACA AGA AGG AGA ATT-3′
NM_001621.5 5′-UUC UCC UUC UUG UAA GUU CTT-3′
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2.9. Statistical Analysis

GraphPad Prism (version 7.0, San Diego, CA) was employed. Data were expressed as mean ± SEM (n = 3), where n is the number of independent experiments; each independent sample was analyzed in triplicate. Statistical tests were conducted by one-way or two-way analysis of variance (ANOVA) with Bonferroni’s test. In all cases, p < 0.05 was considered statistically significant.

3. Results

3.1. The AhR Pathway Mediates the TCDD-Induced EMT in HKC Cells

The noncytotoxic concentrations of TCDD were determined using CCK-8 assay (Figure S1). Significant alterations in mRNA expression of EMT (E-cadherin, fibronectin and MMP9) and inflammatory markers (IL-6) were observed following 24 or 48 h treatment with TCDD (Figure A–D). After 24 h treatment, the mRNA expression levels of E-cadherin and fibronectin showed an EMT-like trend, including a decrease in E-cadherin expression and an increase in fibronectin expression, while longer treatment periods (i.e., 48 h) caused more pronounced concentration-dependent changes in the transcriptional expression levels. After 48 h of 10–8 mol/L TCDD treatment, the greatest effects on gene expression for E-cadherin (56.19% decrease compared to solvent control) and MMP9 (2.39-fold increase) were observed. The gene expression of fibronectin and IL-6 peaked at 10–9 mol/L TCDD, showing 1.87-fold and 8.27-fold increases, respectively, compared to the solvent controls. We then used wound healing assays to test cell migration capacity, which is one of the hallmark alterations in cellular function in EMT. We found that TCDD treatment significantly increased the cell migration distance by 10.09%, 15.34%, and 37.77%, respectively, with 10–10, 10–9, and 10–8 mol/L TCDD treatments compared to the solvent control (Figure E), indicating enhanced EMT progression. These consistent changes at the molecular and cellular levels demonstrate the effect of TCDD to induce EMT in HKC cells.

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TCDD-induced EMT in HKC cells. (A–D) mRNA expression levels of (A) E-cadherin, (B) fibronectin, (C) MMP9, and (D) IL-6 in HKC cells after 24 and 48 h treatment with TCDD (10–10–10–8 mol/L) or 0.1% DMSO. The mRNA expression was determined by qPCR analysis. GAPDH was used as an internal control for quantification. (E) Wound healing assay was used to evaluate the migration distance in response to the treatment with TCDD (10–10–10–8 mol/L) or 0.1% DMSO. Representative images and distances of migration are shown. The images were taken at 0, 24, and 48 h after the scratches (scale bar = 0.5 mm). Values are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the solvent control.

To investigate AhR signaling pathway activation, the mRNA expression levels of its downstream genes was analyzed. Time- and concentration-dependent transcriptional regulations were observed. The level of AhR mRNA exhibited a small but significant reduction, while the downstream genes, including AhRR, CYP1A1, and CYP1B1, showed pronounced upregulation. The maximal effects occurred at 10–8 mol/L TCDD treatment for 48 h, where AhRR, CYP1A1, and CYP1B1 increased by 10.26-fold, 43.34-fold, and 5.28-fold, respectively, compared with the solvent control (Figure A–D). These data confirmed that TCDD induced AhR pathway activation in HKC cells. To further reveal the role of AhR pathway in TCDD-induced EMT, the specific antagonist CH223191 (10–5 mol/L) was employed. The results showed that pretreatment with CH223191 for 3 h prior to the TCDD treatment effectively reversed the transcriptional changes in AhRR, CYP1A1, and CYP1B1 expression upon the 48 h treatment with TCDD at 10–8 mol/L (Figure S2), confirming the function of the AhR pathway antagonist. Crucially, the CH223191 pretreatment only partially reversed the effects on the expression of EMT and inflammatory markers by TCDD (TCDD-only group) in which the TCDD-induced decrease in E-cadherin mRNA expression was restored from 51.07% to 32.64% of the solvent control and the induction of IL-6 mRNA expression was decreased from 8.19-fold to 2.33-fold of the control; regarding the mRNA expression of fibronectin and MMP9, the TCDD-induced upregulations were almost totally reversed by the CH223191 pretreatment (Figure E–H). These results demonstrate that AhR-direct regulation was partially involved in the TCDD-induced expression of the EMT-related marker gene and that additional AhR-indirect mechanisms were also involved.

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AhR is involved in TCDD-induced EMT in HKC cells. (A–D) TCDD activated the AhR signaling pathway in HKC cells. The mRNA expression levels of the AhR signaling pathway downstream genes (A) AhR, (B) AhRR, (C) CYP1A1, and (D) CYP1B1 after 24 and 48 h treatment with TCDD (10–10–10–8 mol/L) or 0.1% DMSO are shown. (E–H) AhR antagonist inhibited TCDD-induced EMT in HKC cells. The mRNA expression of (E) E-cadherin, (F) fibronectin, (G) MMP9, and (H) IL-6 in HKC cells after 48 h treatment with 10–8 mol/L TCDD or 0.1% DMSO together with or without 3 h pretreatment with 10–5 mol/L CH223191 is shown . The mRNA expression was determined by qPCR analysis. GAPDH was used as an internal control for quantification. Values are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the solvent control.

3.2. Molecular Basis of the TCDD-Induced EMT in HKC Cells Based on RNA-Seq Analysis

To further systematically investigate the potential molecular mechanisms underlying the TCDD-induced EMT, RNA-seq analysis was performed on HKC cells treated with 10–8 mol/L TCDD for 24 or 48 h. DEGs were identified using a p adjust cutoff of <0.05 and absolute fold change cutoffs of >1.5 (upregulated) and <0.67 (downregulated) compared with the solvent control. We detected 658 DEGs (390 upregulated, 268 downregulated) at 24 h and 962 DEGs (700 upregulated, 262 downregulated) at 48 h. Notably, upregulated genes predominated over downregulated genes at both time points (Figure ). Further analysis showed that 393 DEGs were common to both time points, 265 DEGs exhibited specific expression changes at 24 h, and 569 DEGs exhibited specific expression changes at 48 h. These transcriptomic changes suggest the progressive activation of TCDD-responsive pathways during TCDD treatment. Importantly, the expression trends of the above-mentioned EMT and inflammatory markers and AhR downstream genes identified by RNA-seq aligned with the qPCR results (Table ). This consistency indicates the reliability of the transcriptomic results, particularly the activation of the AhR signaling pathway and induction of EMT by the TCDD treatments.

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DEGs in HKC cells treatment with TCDD (10–8 mol/L) for (A) 24 h and (B) 48 h are shown by volcano plots. The red dots and blue dots indicate genes that are significantly upregulated and downregulated, respectively. The cutoff was set as <0.05 for p adjust and >1.5 (upregulated) or <0.67 (downregulated) for absolute fold change. Values are compared with the solvent control.

3. RNA-Seq Results of EMT and Inflammatory Markers and AhR Downstream Genes.

gene ID gene name FC (24 h) FC (48 h) regulate
ENSG00000063438 AhRR 10.71 27.79 up
ENSG00000140465 CYP1A1 192.24 265.59 up
ENSG00000138061 CYP1B1 2.48 3.84 up
ENSG00000039068 E-cadherin – 0.52 down
ENSG00000115414 fibronectin – 1.53 up
ENSG00000136244 IL-6 6.70 9.47 up
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Functional annotation and enrichment analyses were performed using three different approaches, namely, KEGG, GO, and Reactome, to characterize the TCDD-induced DEGs. The top 30 significantly enriched terms/pathways (p adjust < 0.05) from each of the six analytical categories (annotation and enrichment analyses per database) were systematically displayed (Figures S3–S8). Fibrosis-related terms/pathways were consolidated into five functional clusters (Adhesion, Proliferation/Cell Growth, Motility/Migration, Differentiation/Morphogenesis, and ECM) and three signaling pathways (MAPK, EGFR, and PI3K) (Tables S1–S12). The chord diagrams revealed network connectivity among these fibrosis-related clusters. The ribbon width represented common DEGs, and the string length indicated total genes per cluster. Upon the 24 h TCDD treatment (Figure A), the “ECM” cluster (168 DEGs), “Proliferation” cluster (121 DEGs), and “Motility” cluster (77 DEGs) (Table S13) were the top three clusters with both the highest DEG counts and strongest intercluster connectivity. Upon the 48 h TCDD treatment (Figure B), the predominant clusters shifted to “ECM” (231 DEGs), “Migration” (119 DEGs), and “Adhesion” (106 DEGs) (Table S14). A multicriteria screening strategy involving fold change, cluster/pathway recurrence frequency, and fibrosis functional relevance were employed to prioritize the core genes for the TCDD-induced EMT, including AREG, EREG, fibrillin-1 (FBN1), C–C motif chemokine ligand 20 (CCL20), TGFA, IL-6, and E-cadherin (Figure C,D), suggesting their central roles in TCDD-induced EMT.

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(A, B) Chord diagrams visualizing the relationships among enriched functional clusters/pathways derived from DEGs associated with fibrosis following (A) 24 h and (B) 48 h TCDD treatment (10–8 mol/L). Strings and color ribbons represent individual enriched clusters and pathways and common genes between cluster and pathway pairs, respectively. The string length represents the total number of genes for the corresponding cluster/pathway, while the ribbon width represents the number of common genes among the paired clusters/pathways. (C, D) Chord diagrams visualizing the relationships between enriched functional clusters/pathways and the screened DEGs following (C) 24 h and (D) 48 h TCDD treatment.

Among the aforementioned seven prioritized core genes, the transcriptomic data for E-cadherin and IL-6 genes were validated in Figure by qPCR analysis. Further qPCR validation for the remaining five core genes revealed regulation by TCDD. The expression of AREG, EREG, and FBN1 exhibited both time- and concentration-dependent upregulation, peaking at 38.68-fold, 16.78-fold, and 31.46-fold increases, respectively, upon treatment with 10–8 mol/L TCDD for 48 h (Figure A,B,D), whereas TGFA and CCL20 showed concentration-dependent responses with maximal increases of 5.18-fold and 1.76-fold upon 24 h TCDD treatments at 10–8 mol/L, respectively (Figure C,E). The qPCR validation results were consistent with the RNA-seq data (Table ).

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Verification of part of the fibrosis-related genes from the RNA-seq results. The mRNA expression of (A) AREG, (B) EREG, (C) TGFA, (D) FBN1, and (E) CCL20 in HKC cells after 24 and 48 h treatment with TCDD (10–10–10–8 mol/L). The mRNA expression was determined by qPCR analysis. GAPDH was used as an internal control for quantification. Values were expressed as mean ± SEM (n = 3). *p < 0.05, ***p < 0.001 compared with the solvent control.

4. Information on RNA-Seq Screened Genes.

gene ID gene name FC (24 h) FC (48 h) regulate
ENSG00000109321 AREG 8.29 10.47 up
ENSG00000124882 EREG 5.37 7.22 up
ENSG00000163235 TGFA 1.93 1.85 up
ENSG00000166147 FBN1 4.36 9.93 up
ENSG00000115009 CCL20 3.17 3.11 up
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3.3. Roles of TCDD-Induced AhR Activation in the Transcriptional Regulation of EMT/Fibrosis-Related Genes

After the effects of TCDD were confirmed on the expression of the core genes, AhR pathway dependence was further assessed by the AhR antagonist CH223191 (10–5 mol/L, 3 h pretreatment). The results showed that AhR pathway blocking by CH223191 differentially modulated the responses to TCDD: the expression of AREG and TGFA paradoxically increased by 1.45-fold and 1.46-fold versus the TCDD-only control (Figure A,C), whereas EREG and CCL20 showed partial (28.62%) and near-complete (96.25%) reversal effects, respectively (Figure B,E), and FBN1 remained unaffected (Figure D). Given these differential effects of the AhR pathway blockade on TCDD-induced gene expression, we therefore sought to determine whether the observed AhR dependence was correlated with the presence of canonical dioxin response elements (DREs; core motifs 5′-TNGCGTG-3′) in the promoter regions of these genes. The promoter analysis of genomic sequences (JASPAR database: https://jaspar.elixir.no/) identified three to four DREs within 2000 bp upstream regions of AREG, EREG, TGFA, and CCL20 but none in FBN1 (Table S15). This presence/absence of the putative DREs on EREG, CCL20, and FBN1 aligns with observed AhR-direct/indirect transcriptional regulations found upon TCDD treatment. Thus, these findings suggest that AhR may mediate transcriptional regulation of EREG and CCL20 through direct DRE binding in TCDD-induced EMT, while FBN1 may be modulated through AhR-indirect mechanisms.

6.

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Effects of the AhR pathway antagonist on part of fibrosis-related genes from the RNA-seq results. The mRNA expression of (A) AREG, (B) EREG, (C) TGFA, (D) FBN1 and (E) CCL20 in HKC cells after 48 h treatment with 10–8 mol/L TCDD or 0.1% DMSO together with or without 3 h pretreatment 10–5 mol/L CH223191 is shown. The mRNA expression was determined by qPCR analysis. GAPDH was used as an internal control for quantification. Values are expressed as mean ± SEM (n = 3). ***p < 0.001 compared with the solvent control. #p < 0.05, ###p < 0.001 compared with TCDD treatment without CH223191.

3.4. The Nephrotoxic Phytochemical AL-I Induces Upregulation of the EMT and Inflammation Marker Genes Primarily via AhR

We have demonstrated that AhR is involved in TCDD-induced EMT in HKC cells and directly or indirectly mediates the expression of EMT and inflammatory markers as well as other EMT-related genes. To investigate the broader regulatory role of the AhR pathway in drug-induced fibrosis, we first evaluated seven AAAs (Table S16) for the AhR activation effect using a luciferase reporter gene assay. Non-cytotoxic concentrations of the selected AAAs were determined by CCK-8 assay (Figure S1). AL-I was demonstrated to have a good concentration-dependent AhR activation effect, prompting its selection for the following mechanistic studies (Figure S10). The results of qPCR analysis revealed AL-I-induced transcriptional regulation of AhR pathway components, including concentration-dependent upregulation of the downstream genes (AhRR, CYP1A1, CYP1B1) and slight upregulation of AhR transcription. Notably, CYP1B1 displayed a time-dependent increase upon AL-I treatment, whereas the effects of the 24 h treatment on other genes were maintained to 48 h (Figure A,C,E,G). The AL-I-induced upregulations of AhRR and CYP1A1 were substantially reversed by AhR siRNA transfection, though CYP1B1 showed little changes (Figure B,D,F,H). The results showed diverse AhR pathway dependences of the AL-I-induced gene expression. Meanwhile, treatment with 10–6 mol/L AL-I significantly reduced the E-cadherin mRNA expression level by a maximum of 31.71%, while fibronectin, MMP9, and IL-6 levels increased by up to 1.68-fold, 1.50-fold, and 2.42-fold, respectively. Furthermore, AhR siRNA transfection reversed these AL-I-induced expression changes (Figure A–D). These results demonstrate the critical involvement of the AhR pathway in AL-I-induced EMT in HKC cells.

7.

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AL-I activated the AhR signaling pathway in HKC cells. The mRNA expression levels of (A) AhR, (C) AhRR, (E) CYP1A1, and (G) CYP1B1 in HKC cells after 24 and 48 h treatment with AL-I (10–8–10–6 mol/L) or 0.1% DMSO and (B) AhR, (D) AhRR, (F) CYP1A1, and (H) CYP1B1 in HKC cells after 48 h treatment with 10–6 mol/L AL-I or 0.1% DMSO before and after AhR knockdown are shown. The mRNA expression was determined by qPCR analysis. GAPDH was used as an internal control for quantification. Values are expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the solvent control.

8.

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AhR is involved in AL-I-induced EMT. The mRNA expression levels of (A) E-cadherin, (B) fibronectin, (C) MMP9, and (D) IL-6 in HKC cells after 48 h treatment with AL-I (10–6 mol/L) or 0.1% DMSO before and after AhR knockdown is shown. The mRNA expression was determined by qPCR analysis. GAPDH was used as an internal control for quantification. Values were expressed as mean ± SEM (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 compared with the solvent control.

4. Discussion

In this study, we employed in vitro experiments to demonstrate that the activation of the AhR signaling pathway plays a critical role in tubulointerstitial fibrosis. We found that AhR activation induced by its potent exogenous ligand TCDD mediates changes in EMT and inflammatory markers via AhR-direct mechanisms in HKC cells. Transcriptome sequencing further deepened our understanding of the regulatory network underlying AhR-mediated EMT/fibrosis and screened out effector genes such as AREG and EREG as well as effector pathways including EGFR and MAPK. We propose that while AhR directly regulates certain EMT and inflammatory markers through binding to DREs, it may also indirectly promote EMT/fibrosis by inducing the expression of EGFR ligands (AREG, EREG, and TGF-α). This suggests the existence of potential AhR-EGFR signaling crosstalk in HKC cells. Moreover, we confirmed that the AhR-direct effects of these profibrotic effects extended beyond the classic environmental pollutant TCDD. The natural nephrotoxin AL-I also activated AhR and induced the expression of EMT and inflammatory markers in HKC cells, and these effects were reversed by AhR knockout. These findings indicated that AhR may serve as a common mediator in renal EMT/fibrosis induced by both POPs and natural nephrotoxins. Therefore, we established the AhR-EMT/inflammation-related profibrotic effect detection system, which can provide support for nephrotoxicity screening and profibrotic risk assessment of various AhR-active pollutants and natural nephrotoxins, while the AhR-EGFR crosstalk mechanism can offer novel mechanistic insights into understanding the pathogenesis of toxin-induced renal fibrosis and formulating targeted therapeutic or preventive strategies for nephrotoxicity.

Aryl hydrocarbon receptor, a high-affinity receptor for TCDD and dioxin-like compounds, mediates toxic responses through nuclear translocation and subsequent regulation of downstream targets such as CYP1A1 and CYP1B1. , Emerging evidence reveals the complex role of AhR in fibrosis regulation across multiple organs. For instance, cardiomyocyte-specific AhR knockout mice (βMhc:cre; Ahrfx/fx) and the AhR antagonist CH223191 significantly inhibited cardiac and pulmonary fibrosis progression induced by TCDD and bleomycin (BLM). These were supported by reduced histopathological evidence and collagen deposition. , In contrast, studies in liver tissue reported different findings: compared with controls, Orlowska et al. and Patil et al. observed marked upregulation of fibrotic marker genes in both TCDD-exposed DRE-binding-deficient mice (PkmΔDRE) and hepatocyte-specific AhR knockout mice (AHR-HKO) fed a high-fat, high-fructose, high-cholesterol diet. Moreover, in renal fibrosis research, Chen et al. and Xie et al. observed that AhR inhibition by siRNA and CH223191 significantly reversed IS-induced upregulation of IL-6 and fibronectin expression and downregulation of E-cadherin in human proximal tubular epithelial cells (HK-2) and mouse tubular epithelial cells (mTECs), which is consistent with our results. Han et al. previously suggested that TCDD induced hepatic fibrosis in C57BL/6 mice via activation of Akt and NF-κB signaling pathways, which provided mechanistic insight into AhR-indirect fibrosis processes induced by TCDD. Therefore, we performed RNA-seq to further systematically investigate potential mechanisms of TCDD-induced AhR-indirect fibrosis.

Based on the potential effector genes screened by RNA-seq and multiple (KEGG, GO, and Reactome) enrichment analysis results, the EGFR pathway has attracted our attention. A number of studies have shown a close association between the EGFR signaling pathway and fibrosis. Wang et al. and Sangamish et al. revealed that procyanidin C1 (PCC1) and butyrate can inhibit BLM-induced skin and pulmonary fibrosis in male C57BL/6J mice and Swiss albino mice by targeting EGFR. , Similarly, Sun et al. also showed that MMP10 promotes renal fibrosis in both HK-2 cells and unilateral ureteral obstruction (UUO) mice through EGFR-mediated β-catenin transactivation. In addition, EGFR ligands also play a significant role in fibrosis. For example, AREG promotes renal and pulmonary fibrosis by inducing the expression of fibrosis markers such as fibronectin and E-cadherin in both proximal tubule cell-specific a disintegrin and metalloproteinase 17 knockout mice (ADAM17 PTC-KO) and human lung epithelial A549 cells. , Wu et al. also demonstrated that EREG drives hepatic fibrosis by stimulating Col1A1 and α-SMA expression in HSCs. These results suggest that both EGFR and its ligands contribute significantly to fibrosis significantly. These results are consistent with those from our experiments. In developing mouse ureters and HK-2 cells, AREG expression was significantly increased upon exposure to TCDD and TGF-β1, , and Choi et al. and Campion et al. also reported that TCDD can increase the secretion and expression of TGFA and AREG in normal human epidermal keratinocytes (NHEKs). , Therefore, we propose that TCDD may induce renal fibrosis by stimulating EGFR ligand expression and activating EGFR in HKC cells. On the other hand, it has been demonstrated that the transcription and translation of AREG and EREG are regulated by AhR in human head and neck squamous cell carcinoma (HNSCC) cell lines and mouse primary mammary tumor cell lines [K14cre BRCA1f/f p53f/f (KBP)]. , Among them, John et al. found that the mRNA expression level of EREG partially decreased after addition of CH223191, and there were DREs in the promoters of AREG and EREG, which are consistent with our results. However, the unexpected increase in AREG expression upon AhR antagonism in HKC cells suggests additional, tissue-specific AhR-indirect regulatory mechanisms, such as MAPK, EGFR, and PI3K-Akt signaling. ,, Accordingly, our findings provide new insights into the mechanisms of TCDD-induced renal fibrosis.

Fibrosis is a common pathological feature of most chronic inflammatory diseases, defined by the excessive accumulation of ECM components such as fibronectin and the secretion of anti-inflammatory mediators. Severe or repetitive injuries may lead to tissue destruction, organ dysfunction, and ultimately organ failure. As a typical persistent organic pollutant, TCDD can accumulate in animal fats and plant tissues, inducing diverse toxic effects on human health, including causing fibrosis of organs and tissues. Beyond TCDD, growing evidence suggests that other classical and emerging environmental pollutants with AhR-activating potential may similarly promote renal fibrosis. For example, studies by Ruan et al. demonstrated that certain PAHs (e.g., phenanthrene) and polychlorinated biphenyls (PCBs) (e.g., PCB138) can also induce renal fibrosis in mice. , Miao et al. provided clinical evidence that that elevated serum PAH metabolites in CKD patients promote renal fibrosis through AhR activation. However, existing research on TCDD-induced nephrotoxicity mainly focuses on the disruption of physiological and pathological indicators, with a particular emphasis on oxidative stress as a potential factor, which is still largely limited in understanding the molecular mechanisms of renal fibrosis and related diseases. In this study, using TCDD as a model pollutant, we have demonstrated the crucial role of the AhR-EMT/inflammation signaling axis in renal fibrosis. The established AhR-EMT/inflammation-related profibrotic effect detection system not only provides a novel perspective for investigating the mechanisms of renal fibrosis induced by pollutants such as TCDD but also suggests that the AhR-EMT/inflammation axis could serve as a biomarker for nephrotoxicity screening and risk assessment of environmental pollutants, particularly emerging pollutants. This method holds significant implications for the prevention of environmental-factor-related CKD.

Aristolactam, a recognized nephrotoxic component of the Aristolochiaceae family, has been well-documented to cause renal injury and diseases. Employing AL-I concentrations slightly exceeding those used in our experiments (2.5 × 10–6 to 1 × 10–5 mol/L) can significantly increase fibronectin and decrease E-cadherin expression in HK-2 cells. Although direct evidence of AhR-mediated AL-I-induced fibrosis is limited, research by Xiao et al. and Ma et al. suggests that the precursor compound of AL-I (aristolochic acid I, AAI) can target AhR and exert toxic effects by activating its downstream genes. Based on the molecular structure similarity between AAI and AL-I and the AhR dependence of AL-I-induced HKC cell fibrosis in our experimental results, appropriate evidence is provided for the inference that AhR is involved in the subsequent transcriptional regulation induced by AL-I. Previous studies on AL-I have primarily focused on its DNA adduct formation, driving genotoxic and carcinogenic effects. Our findings reveal that AhR activation and its mediated non-genotoxic pathways (e.g., promoting EMT and inflammation) are also core mechanisms underlying AL-I-induced tubulointerstitial injury and renal EMT/fibrosis. It provides a foundation for developing targeted interventions against aristolochic acid nephropathy (AAN) and similar AhR-mediated renal injuries. Furthermore, AhR activation and its associated early molecular events (e.g., EMT and inflammatory marker alterations) may serve as sensitive biomarkers. These biomarkers may be used to monitor the risk of early renal injury and fibrosis in populations exposed to aristolochic acid-containing herbs or other environmental AhR agonists, enabling early warning and timely intervention. Interestingly, studies by Wang et al. found that the natural compound acteoside attenuated renal fibrosis by alleviating inflammation via suppressing AhR signaling. This finding further supports the important role of AhR in renal fibrosis and highlights its potential as a promising therapeutic target. Therefore, future efforts to develop specific AhR antagonists or identify novel natural AhR inhibitors from herbal compounds could offer novel therapeutic strategies for AAN and other AhR-mediated kidney diseases. This study has limitations that warrant consideration, as the results are primarily derived from in vitro HKC cell models and focus largely on transcriptional-level analyses. To strengthen the validity of these observations, future studies should employ cotreatment models or in vivo systems to verify their pathophysiological relevance and perform multilevel assessments, including protein and cellular levels, for a more comprehensive understanding of the underlying mechanisms. Furthermore, direct evidence for AhR-EGFR crosstalk could be strengthened by detecting the phosphorylation status of EGFR and its downstream effectors, such as ERK and AKT.

5. Conclusion

This study demonstrates that AhR activation by TCDD or AL-I induces EMT and inflammatory responses in HKC cells, promoting a profibrotic phenotype. Transcriptomic analysis revealed the activated AhR-directed upregulation of EGFR ligands, suggesting crosstalk between AhR and EGFR signaling pathways. Thus, we established an AhR-EMT/inflammation-related profibrotic effect detection system for screening and assessing the nephrotoxic potential of compounds with AhR activity. Our findings also indicate that AhR may serve as a common mediator in diverse nephrotoxin-induced renal injury. However, these in vitro observations require further in vivo validation to fully elucidate their pathophysiological relevance.

Supplementary Material

eh5c00494_si_001.pdf (2.1MB, pdf)

Acknowledgments

This work was supported by the National Natural Science Foundation of China (22021003), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0750300), and Chongqing Natural Science Foundation General Project (CSTB2024NSCQ-KJFZMSX0029).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/envhealth.5c00494.

  • Details of RNA-seq analysis, prediction of DREs of RNA-seq screened genes, aristolochic acid analogues information, cell viability assay, RT-qPCR analysis, ELISA, and AhR activation assay (PDF)

The authors declare no competing financial interest.

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