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. 2026 Jul 10;17:8600. doi: 10.1038/s41467-026-75114-5

Uremic toxins promote renal fatty acid synthesis and fibrosis via activating aryl hydrocarbon receptor

Hongyan Xie 1, Yitao Zhang 2, Li Lu 2,3, Hengjiang Guo 2, Xi’ang Sun 2, Ming Ning 2, Xin Xie 2, Luxin Li 2, Yangyang Niu 1, Jingyao Li 2, Xin Wang 2, Lehao Wu 1, Li Zhou 2, Wei Zhang 2, Yingying Zhang 1, Chen Xu 2,✉, Limin Lu 2,4,5,✉, Chen Yu 1,✉
PMCID: PMC13486649  PMID: 42431865

Abstract

Chronic kidney disease (CKD) affect about 10% of adults worldwide, with dyslipidemia being a common feature. Abnormalities in renal lipid metabolism have been strongly implicated in CKD progression; however, the mechanisms by which CKD leads to lipid metabolism disturbances remain underexplored. Here we show that following the accumulation of uremic toxins, the synthesis and deposition of lipids, along with the uremic toxin receptor aryl hydrocarbon receptor (AhR), are upregulated in the kidneys. Tubule-specific AhR knockout in male mice alleviates uremic toxin-induced increases in renal fatty acid (FA) synthesis, lipid accumulation and fibrosis. Immunoprecipitation‒mass spectrometry identifies nuclear receptor subfamily 1 group D member 1 (NR1D1) as an AhR-interacting protein. Co-immunoprecipitation confirms that AhR interacts with NR1D1 and promotes its ubiquitin-mediated degradation. As NR1D1 is an FA synthesis suppressor, its reduction relieves the transcriptional repressing effects on sterol regulatory element-binding protein 1 (SREBP1), thereby enhancing SREBP1/fatty acid synthase (FASN) pathway activity and FA synthesis. In summary, by acting on AhR, the accumulation of uremic toxins may accelerate renal fibrosis via the SREBP1/FASN pathway-mediated increase in FA synthesis.

Subject terms: Chronic kidney disease, Renal fibrosis

Abnormalities in renal lipid metabolism are linked to CKD progression, but how CKD drives lipid disturbances remains unclear. Here, the authors show that by acting on AhR, the accumulation of uremic toxins may accelerate renal fibrosis via the SREBP1/FASN pathway-mediated increase in FA synthesis

Introduction

Chronic kidney disease (CKD) is a global health issue, with a prevalence of about 10% worldwide1. Although lipid levels in healthy kidneys are generally low, lipids are a major energy source of the kidney, especially in proximal tubular epithelial cells (TECs), where the adenosine triphosphate (ATP) demand is relatively high for reabsorptive and secretory processes2. It has been noticed that renal lipid metabolism is disturbed in CKD patients, and the disturbances in lipid metabolism are implicated in renal deterioration3. However, how renal dysfunction leads to disturbances in lipid metabolism has received limited attention.

The kidney serves as the main organ that eliminates end metabolic wastes from body fluids. Following the decline in kidney function, the metabolic wastes of body cells and gut microbiota, such as indoxyl sulfate (IS), are retained in the body fluids of patients with CKD. These metabolic wastes are also known as uremic toxins4. The accumulation of uremic toxins exerts deleterious effects on multiple tissues and organs, but the exact mechanisms have long been ignored.

Following the identification of aryl hydrocarbon receptor (AhR) as the endogenous receptor for multiple uremic toxins, the investigations into the impact of uremic toxins on body tissues and organs are gaining increasing attention5. AhR was originally identified as a sensor that responds to environmental pollutants6. Subsequent research revealed that multiple uremic toxins are the endogenous ligands of AhR7. AhR was first identified to act as a ligand-activated transcription factor8; Shigeaki Kato et al. demonstrated that AhR can also act as an adaptor for the cullin 4B E3 ligase complex to regulate the ubiquitination and degradation of target proteins9.

Studies have shown that the activation of AhR by uremic toxins contributes to disease progression. For example, the activation of AhR promotes smooth muscle cell osteogenic reprogramming and vascular calcification10, facilitates colorectal cancer development11, suppresses postischemic angiogenesis12, and accelerates renal senescence13.

In this work, we show that the accumulation of uremic toxins leads to renal lipid metabolic disturbances by acting on AhR. The upregulation and activation of AhR by uremic toxins facilitate the degradation of the transcriptional repressor nuclear receptor subfamily 1 group D member 1 (NR1D1) via its E3 ubiquitin ligase activity. This, in turn, increases the activity of transcription factor sterol regulatory element-binding protein 1 (SREBP1 or Srebp1) and upregulates the expression of its downstream target, fatty acid synthase (FASN). This cascade enhances fatty acid (FA) synthesis and lipid accumulation, ultimately contributing to renal fibrosis.

Results

The uremic toxin IS promoted FA synthesis and lipid accumulation in fibrotic kidneys

The analysis of the published data revealed that the serum triglyceride (TG) and IS levels were increased in patients with CKD (Fig. 1a, b)14. Pathway analysis of the published RNA-seq data from ischemia reperfusion (IR)-induced CKD mouse kidneys (GSE98622) revealed that the differentially expressed genes were enriched in lipid metabolism-associated pathways (Fig. 1c). The mRNA levels of the FA synthesis-associated genes were consistently increased and positively correlated with renal fibrosis markers, including fibronectin (Fn) and collagen 1a1 (Col1a1, Fig. 1d, e), while the changes in the FA oxidation- and transportation-associated genes were unordered and the correlations of those genes with renal fibrosis were uncertain (Supplementary Fig. 1a, b). So this study aimed to investigate how uremic toxins lead to changes in renal FA synthesis.

Fig. 1. Elevated uremic toxin IS promoted FA synthesis and lipid accumulation during renal fibrosis.

Fig. 1

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a The relative intensity of five kinds of serum TG in normal controls and patients with stage 1–5 CKD (Normal, n = 116; CKD1, n = 120; CKD2, n = 104; CKD3, n = 110; CKD4, n = 119; CKD5, n = 134). b The relative intensity of serum IS (Normal, n = 116; CKD1, n = 120; CKD2, n = 104; CKD3, n = 110; CKD4, n = 119; CKD5, n = 134). c Gene Ontology (GO) pathways associated with lipid metabolism of differentially expressed genes (n = 3). d Heatmap of FA synthesis-associated genes in the kidneys of control and IR-induced CKD mice (n = 3). e Correlation plot showing Spearman correlation values between the mRNA levels of FA synthesis-associated genes and fibrosis-associated genes in the kidneys (n = 6). f Experimental design of the IR animal model. g Serum total IS, creatinine and BUN levels in sham- or IR-treated mice (n = 6). h HE, Masson and Oil Red O staining of kidneys from sham- or IR-treated mice (n = 6 mice). Scale bar = 50 μm. i Western blot and quantification showing the protein levels of SREBP1 and FASN in the kidneys (n = 6). j Western blot and quantification showing the protein levels of FN and α-SMA in the kidneys (n = 6). k Western blot and quantification showing the protein levels of SREBP1 and FASN in mTECs at the indicated time points after IS treatment (n = 3 independent biological replicates). l Oil Red O staining and quantification showing lipid accumulation in mTECs (n = 3 independent biological replicates). Scale bar = 50 μm. m Western blot and quantification showing the protein levels of FN and α-SMA in mTECs (n = 3 independent biological replicates). Data were shown as mean ± SD. Statistical analysis was performed by one‑sided Fisher’s exact test with Benjamini‑Hochberg correction for multiple comparisons (c), two-tailed unpaired Student’s t-test (the middle and right panels of g, j), two-tailed unpaired Welch’s t-test (the left panel of g, i), and one-way ANOVA with Tukey’s multiple comparisons test (k–m). Source data are provided as a Source Data file.

To further identify the association between uremic toxins and FA synthesis, an IR-induced renal fibrosis model was constructed. The serum IS, creatinine and blood urea nitrogen (BUN) levels were increased in IR-treated mice (Fig. 1f, g). Hematoxylin-eosin (HE), Masson and Oil Red O staining indicated that the kidneys of IR-treated mice displayed obvious tubular atrophy, interstitial fibrosis and lipid accumulation (Fig. 1h). Western blot revealed that SREBP1, the key regulator of FA synthesis, and FASN, the downstream target of SREBP1 and rate-limiting enzyme in FA synthesis, were significantly increased in the kidneys of IR-treated mice (Fig. 1i). The fibrosis markers FN and α-smooth muscle actin (α-SMA) were also increased (Fig. 1j). To confirm the results observed in IR-treated mice, an IS gavage-induced renal fibrosis model was constructed (Supplementary Fig. 2a). Compared with vehicle-treated mice, IS-treated mice showed higher serum IS levels, but comparable levels of serum creatinine and BUN (Supplementary Fig. 2b). Similar to IR-treated mice, IS-treated mice exhibited significant renal tubular injury, interstitial fibrosis and lipid accumulation, as well as increases in SREBP1, FASN, FN and α-SMA (Supplementary Fig. 2c–e). In cultured mouse tubular epithelial cells (mTECs), IS induced increases in SREBP1 and FASN expression, lipid accumulation and fibrotic phenotype changes (Fig. 1k–m). The data suggest that the accumulation of uremic toxins stimulates FA synthesis and lipid accumulation in TECs, which may aggravate renal injury and fibrosis.

The uremic toxin receptor AhR was associated with FA synthesis during renal fibrosis

AhR is the endogenous receptor of uremic toxins. Reanalysis of published kidney RNA-seq data from CKD mice (GSE98622) and patients (GSE175759) revealed elevated AhR mRNA levels in the kidneys of IR-induced CKD mice and CKD patients (Fig. 2a). AhR mRNA levels were negatively correlated with estimated glomerular filtration rate (eGFR) and positively correlated with interstitial fibrosis (Fig. 2b). AhR mRNA levels were positively correlated with FA synthesis-associated genes, whereas the correlations between AhR and FA oxidation- or transportation-associated genes were indeterminate (Fig. 2c, and Supplementary Fig. 3a, b). Immunohistochemistry (IHC) and Western blot results showed that AhR was upregulated in the kidneys of IR- or IS-treated mice and was mainly distributed in renal tubules (Fig. 2d, e). In addition, the increase in AhR was observed in IS-treated mTECs in a concentration- and time-dependent manner (Fig. 2f, g). IHC of residual kidney biopsy samples from patients with various kidney diseases, including minimal change disease (MCD), diabetic nephropathy (DN), IgA nephropathy (IgAN) and anti-glomerular basement membrane glomerulonephritis (anti-GBM GN), also showed that AhR was increased when compared with para-carcinoma kidney tissues and mainly located in TECs (Fig. 2h).

Fig. 2. The uremic toxin receptor AhR was increased and positively correlated with FA synthesis in fibrotic kidneys.

Fig. 2

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a The mRNA levels of AhR in the kidneys of control (n = 3) and IR-induced CKD (n = 3) mice from the GSE98622 mouse dataset, or healthy controls (n = 15) and patients with CKD (n = 26) from the GSE175759 human dataset. b Correlation plots showing Spearman correlation values between renal AhR mRNA and eGFR (n = 41) or interstitial fibrosis (n = 24). c Correlation plots showing Spearman correlation values between the mRNA levels of AhR and FA synthesis-associated genes in the kidneys from the GSE98622 mouse dataset (n = 6) and the GSE175759 human dataset (n = 41). d, e IHC staining, and Western blot and quantification showing AhR expression in the kidneys of sham- or IR-treated mice (d), and vehicle- or IS-treated mice (e) (n = 6). Scale bar = 50 μm. f, g Western blot and quantification showing the protein levels of AhR in mTECs at the indicated concentration of IS (f) or time after IS treatment (g) (n = 3 independent biological replicates). h IHC staining showing AhR expression in kidney biopsy samples from para-carcinoma kidney tissues (used as healthy control), as well as from patients with various kidney diseases, including minimal change disease (MCD), diabetic nephropathy (DN), IgA nephropathy (IgAN) and anti-glomerular basement membrane glomerulonephritis (anti-GBM GN) (n = 1 independent biological replicate). Scale bar = 25 μm. Data were shown as mean ± SD. Statistical analysis was performed by two-tailed unpaired Welch’s t-test (the right panel of a), two-tailed test (b), two-tailed unpaired Student’s t-test (d, e) and one-way ANOVA with Tukey’s multiple comparisons test (f, g). Source data are provided as a Source Data file.

Elevated AhR promoted FA synthesis, lipid accumulation and fibrotic phenotype transition in cells

AhR expression in mTECs was knocked down using small interfering RNA (siRNA) (Supplementary Fig. 4a). Oil Red O staining indicated that AhR knockdown attenuated lipid accumulation in IS-treated mTECs (Fig. 3a). IS-induced increases in SREBP1 and FASN at both the protein and mRNA levels were also inhibited by AhR knockdown (Fig. 3b, c). Besides, the IS-induced increases in cellular TG and free fatty acid (FFA) contents were also suppressed by AhR knockdown (Fig. 3d). Glucose serves as a key substrate for de novo FA synthesis15. Using [U-¹³C]glucose to trace FA synthesis, we found that IS treatment significantly increased the incorporation of ¹³C from glucose into palmitic acid and stearic acid, and AhR knockdown attenuated the changes (Fig. 3e, and Supplementary Fig. 4b, c). These results confirmed that the accumulated lipids in uremic toxin-treated TECs were derived from de novo lipid synthesis pathway and that AhR promoted lipid accumulation by increasing FA synthesis. To investigate whether AhR promoted lipid accumulation by regulating FA degradation, we assessed key markers of FA oxidation (PPARα, CPT1A and ACOX1) by Western blot. AhR knockdown or knockout attenuated decreases in PPARα, CPT1A and ACOX1 in IS-treated mTECs, and in the kidneys of IR and IS mice (Supplementary Fig. 4d–f). Oxygen consumption rate (OCR) of mTECs was also assessed by Seahorse. The results showed that IS treatment reduced both basal and maximal OCR, and decreased ATP production, which were attenuated by AhR knockdown (Supplementary Fig. 4g). These results indicated that AhR also promoted lipid accumulation by inhibiting FA oxidation. In addition, cell injury can activate intracellular phospholipases, such as cytoplasmic phospholipase A2 (PLA2), which mediate phospholipolysis16. This process leads to partial phospholipid depletion and increases in FFAs and lysophospholipids. To investigate whether AhR promoted lipid accumulation by regulating phospholipolysis, we assessed PLA2 activity. The results showed that PLA2 activity was unchanged in IS-treated cells, or in the kidneys of IR and IS mice. And knockdown or knockout of AhR in tubules did not affect PLA2 activity (Supplementary Fig. 4h). These results indicated that phospholipolysis was not an important contributor to lipid formation following the accumulation of uremic toxins. Furthermore, the IS-induced increases in FN and α-SMA were also suppressed by AhR knockdown (Fig. 3f).

Fig. 3. AhR promoted FA synthesis, lipid accumulation and fibrotic phenotype transition in mTECs and primary cultured TECs.

Fig. 3

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mTECs were transfected with AhR small interfering RNA (si-AhR) or negative control siRNA (si-NC) for 12 h, followed by treatment with or without IS for an additional 36 h. a Oil Red O staining and quantification showing lipid accumulation in mTECs (n = 3 independent biological replicates). Scale bar = 50 μm. b Western blot and quantification showing the protein levels of SREBP1 and FASN in mTECs (n = 3 independent biological replicates). c qPCR showing the mRNA levels of Srebf1 and Fasn in mTECs (n = 3 independent biological replicates). d Cellular TG and FFA contents in mTECs (n = 3 independent biological replicates). e The percentages of isotopomers of FA 16:0 in mTECs after [U-13C]glucose tracing (n = 3 independent biological replicates). f Western blot and quantification showing the protein levels of FN and α-SMA in mTECs (n = 3 independent biological replicates). mTECs were transfected with an adenovirus containing the Flag-tagged mouse AhR gene (Ad-AhR) or control adenovirus (Ad-NC) for 12 h, followed by treatment with or without IS for an additional 36 h. g Oil Red O staining and quantification in mTECs (n = 3 independent biological replicates). Scale bar = 50 μm. h Western blot and quantification showing the protein levels of SREBP1 and FASN in mTECs (n = 3 independent biological replicates). i qPCR showing the mRNA levels of Srebf1 and Fasn in mTECs (n = 3 independent biological replicates). j Cellular TG and FFA contents in mTECs (n = 3 independent biological replicates). k Western blot and quantification showing the protein levels of FN and α-SMA in mTECs (n = 3 independent biological replicates). l Primary TECs isolated from Cre−/AhRfl/fl or Cre+/AhRfl/fl mice were treated with IS for 36 h. Oil Red O staining and quantification in primary TECs (n = 3 independent biological replicates). Scale bar = 12.5 μm. Data were shown as mean ± SD. Statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparisons test (a–l). Source data are provided as a Source Data file.

AhR was overexpressed in mTECs by transfecting an adenovirus containing the Flag-tagged mouse AhR gene (Ad-AhR, Supplementary Fig. 4i). Overexpression of AhR in IS-treated mTECs exacerbated lipid accumulation and the increases in SREBP1, FASN, TG, FFA, FN and α-SMA (Fig. 3g–k). However, AhR overexpression did not induce those changes in cells without IS stimulation. Primary TECs were successfully isolated from Cre−/AhRfl/fl mice and Cre+/AhRfl/fl mice (Supplementary Fig. 5a, b). The results showed that AhR knockout significantly mitigated the IS-induced increases in lipid accumulation, SREBP1, FASN, TG, FFA, FN and α-SMA, as well as a decrease in NR1D1 (Fig. 3l and Supplementary Fig. 5c–f). These results suggest that AhR mediates uremic toxin-induced FA synthesis, lipid accumulation and fibrotic phenotype transition, probably via the SREBP1/FASN pathway.

AhR promoted the ubiquitination and proteasomal degradation of NR1D1

Immunoprecipitation‒mass spectrometry (IP‒MS) analysis was used to identify the molecules that interact with AhR in AhR-overexpressing mTECs. Among the top 14 proteins identified, NR1D1 was the only molecule associated with cell metabolism (Fig. 4a, and Supplementary Data 1). STRING analysis revealed that NR1D1 was able to interact with AhR (Fig. 4b). Co-immunoprecipitation (Co-IP) results confirmed that AhR interacted with NR1D1 (Fig. 4c). Previous studies reveal that NR1D1 is a pivotal regulator of the SREBP1/FASN pathway17,18. So, NR1D1 was selected as the candidate for further investigation. Structurally, AhR comprises a bHLH domain, two PAS domains, an acidic domain and a Q-rich domain7. Molecular docking analysis suggested that the acidic domain was responsible for the interaction of AhR with NR1D1 (Fig. 4d). So, plasmids encoding Myc-tagged full-length AhR (AhR WT) and the acidic domain-deleted AhR mutant (AhR Δacid) were constructed. Co-IP results showed that AhR WT interacted with NR1D1, whereas the deletion of the acidic domain abolished the interaction (Fig. 4e). As the acidic domain is the region where AhR interacts with the cullin 4B E3 ubiquitin ligase complex9, we speculated that AhR promoted the ubiquitination and proteasomal degradation of NR1D1 via its E3 ubiquitin ligase activity. The mRNA levels of Nr1d1 did not show obvious differences in the kidneys of IR- or IS-treated mice when compared with control mice (Fig. 4f), whereas the protein levels of NR1D1 were significantly decreased (Fig. 4g). Cycloheximide (CHX) was used to inhibit protein synthesis, and the result showed that IS obviously shortened the half-life of NR1D1 (Fig. 4h). The IS-induced decrease in NR1D1 was reversed by treatment with the proteasome inhibitor MG132 (Fig. 4i), which suggested that the shortened half-life of NR1D1 was due to the increase in proteasomal degradation. IS treatment enhanced the ubiquitination of NR1D1, while AhR knockdown inhibited this effect (Fig. 4j). AhR knockdown alleviated the IS-induced decrease in NR1D1 protein levels, and AhR overexpression aggravated this decrease (Fig. 4k, l). In the presence of IS, overexpression of AhR WT promoted the ubiquitination of NR1D1, whereas the AhR mutant without the acidic domain abolished the effect (Fig. 4m). Those data suggest that increased AhR facilitates NR1D1 degradation via its E3 ubiquitin ligase activity. Notably, AhR is also a ligand-activated transcription factor8. To determine whether the transcriptional activity of AhR was involved in regulating NR1D1 ubiquitination, we constructed a Myc-tagged truncated AhR mutant lacking residues 110–119 (AhR Δα), which abolished the transcriptional activity of AhR by inhibiting the nuclear translocation. Co-IP analysis demonstrated that both AhR WT and AhR Δα were able to interact with NR1D1, and that deletion of the Δα domain did not affect NR1D1 ubiquitination following IS treatment. Those results suggest that AhR’s transcriptional activity was not required for the regulation of NR1D1 ubiquitination (Supplementary Fig. 6a, b).

Fig. 4. AhR decreased NR1D1 expression by promoting its ubiquitination and proteasomal degradation.

Fig. 4

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a IP‒MS showing the top 14 proteins that interacted with AhR in mTECs. b STRING analysis showing the protein‒protein interaction network of AhR. c Cell lysates from IS-treated mTECs were immunoprecipitated with anti-IgG, anti-AhR, or anti-NR1D1 antibodies and subsequently analyzed by immunoblotting with the indicated antibodies (n = 3). d The full structure of mouse AhR and the molecular docking model between AhR and NR1D1 proteins. e Schematic of the full-length AhR (AhR WT) and a truncated AhR mutant lacking the acidic domain (AhR Δacid). 293T cells were cotransfected with plasmids encoding Flag-tagged NR1D1, and Myc-tagged AhR WT or AhR Δacid, followed by treatment with IS. Cell lysates were immunoprecipitated with an anti-Flag antibody and subsequently analyzed by immunoblotting with the indicated antibodies (n = 3). f qPCR showing the mRNA levels of Nr1d1 in the kidneys (n = 6 mice). g Western blot and quantification showing NR1D1 expression in the kidneys (n = 6 mice). h Western blot and quantification showing NR1D1 expression in mTECs treated with or without IS or CHX at the indicated time points (n = 3 independent biological replicates). i Western blot and quantification showing NR1D1 expression in mTECs treated with IS for 36 h and with MG132 for 4 h (n = 3 independent biological replicates). j Cell lysates from mTECs transfected with si-AhR and treated with IS and MG132 were immunoprecipitated with an anti-NR1D1 antibody and subsequently analyzed by immunoblotting with the indicated antibodies (n = 3). k, l Western blot and quantification showing NR1D1 expression in mTECs (n = 3 independent biological replicates). m 293T cells were cotransfected with the indicated plasmids, followed by treatment with IS and MG132. Cell lysates were immunoprecipitated with an anti-Flag antibody and subsequently analyzed by immunoblotting with the indicated antibodies (n = 3). Data were shown as mean ± SD. Statistical analysis was performed by two-tailed unpaired Student’s t-test (f, g) and two-way ANOVA with Tukey’s multiple comparisons test (h, i, k, l). Source data are provided as a Source Data file.

AhR acts as an adaptor in the cullin 4B E3 ubiquitin ligase complex9. Co-IP results showed that AhR or NR1D1 interacted with cullin 4B (Fig. 5a). Cullin 4B knockdown inhibited IS-induced increases in cullin 4B and NR1D1 ubiquitination, and attenuated the IS-induced decrease in NR1D1 protein levels (Fig. 5b–d). Those data suggest that AhR promotes the degradation of NR1D1 via the cullin 4B E3 ubiquitin ligase complex.

Fig. 5. AhR promoted the degradation of NR1D1 via the cullin 4B E3 ubiquitin ligase complex.

Fig. 5

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a 293T cells were cotransfected with plasmids encoding Myc-tagged AhR WT or Flag-tagged NR1D1, followed by treatment with IS. Cell lysates were immunoprecipitated with anti-IgG, anti-Myc or anti-Flag antibodies and subsequently analyzed by immunoblotting with the indicated antibodies (n = 3). b mTECs were transfected with cullin 4B small interfering RNA (si-cullin 4B) or negative control siRNA (si-NC) for 12 h, followed by treatment with or without IS for an additional 36 h. Western blot and quantification showing the protein levels of cullin 4B (n = 3 independent biological replicates). c Cell lysates from mTECs transfected with si-cullin 4B and treated with IS and MG132 were immunoprecipitated with an anti-NR1D1 antibody and subsequently analyzed by immunoblotting with the indicated antibodies (n = 3). d Western blot and quantification showing the protein levels of NR1D1 in mTECs (n = 3 independent biological replicates). Data were shown as mean ± SD. Statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparisons test (b, d). Source data are provided as a Source Data file.

Decreased NR1D1 mediated AhR-induced FA synthesis, lipid accumulation and fibrotic phenotype transition by relieving the transcriptional repression on Srebf1

mTECs were treated with the NR1D1 agonist SR900919, and the results showed that SR9009 did not influence the protein levels of NR1D1 (Supplementary Fig. 7a). SR9009 inhibited IS-induced increases in the Oil Red O-positive area, the protein and mRNA levels of SREBP1 and FASN, cellular TG and FFA contents, and the protein levels of FN and α-SMA (Fig. 6a–e). NR1D1 was previously documented as a transcriptional repressor20. The analysis of chromatin immunoprecipitation (ChIP)-seq data of NR1D1 from liver tissues in the ENCODE website revealed that the epigenetic marks (H3K4me3, H3K27ac and RNA Pol II) and NR1D1 were enriched at the promoter region of Srebf1 (Fig. 6f). The NR1D1-binding motif was identified using the MEME Suite (Fig. 6g). ChIP‒PCR and ChIP‒qPCR confirmed that NR1D1 can bind to the predicted region of Srebf1 promoter and this binding was decreased after IS treatment (Fig. 6h, i). These results suggested that the decreased NR1D1 binding to the promoter region of Srebf1 following IS treatment relieved the repression of NR1D1 on the SREBP1/FASN pathway.

Fig. 6. The downregulation of NR1D1 expression induced by AhR promoted FA synthesis and fibrotic phenotype transition by relieving the transcriptional repression on Srebf1 in mTECs.

Fig. 6

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mTECs were pretreated with SR9009 for 1 h, followed by treatment with IS for an additional 36 h. a Oil Red O staining and quantification showing lipid accumulation in mTECs (n = 3 independent biological replicates). Scale bar = 50 μm. b Western blot and quantification showing the protein levels of SREBP1 and FASN (n = 3 independent biological replicates). c qPCR showing the mRNA levels of Srebf1 and Fasn in mTECs (n = 3 independent biological replicates). d Cellular TG and FFA contents in mTECs (n = 3 independent biological replicates). e Western blot and quantification showing the protein levels of FN and α-SMA in mTECs (n = 3 independent biological replicates). f Enrichment of H3K4me1, H3K4me3, H3K27ac, RNA Pol II, and the transcription factor NR1D1 at the promoter region of Srebf1 in mouse livers from the ENCODE website. The binding region is indicated by a gray square. g The NR1D1-binding motif was identified using the MEME Suite (https://meme-suite.org/meme). h, i ChIP‒PCR (h) and ChIP‒qPCR (i) showing the binding of NR1D1 to the predicted binding motif at the promoter region of Srebf1 in mTECs treated with IS for 36 h (n = 3 independent biological replicates). j mTECs were transfected with Myc-tagged AhR WT plasmids or Flag-tagged NR1D1 plasmids, followed by treatment with IS. Oil Red O staining and quantification showing lipid accumulation in mTECs (n = 3 independent biological replicates). Scale bar = 50 μm. Data were shown as mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test (a–e, j), and two-way ANOVA with Tukey’s multiple comparisons test (i). Source data are provided as a Source Data file.

To further clarify the role of NR1D1 in AhR-mediated FA synthesis following IS treatment, mTECs were transfected with plasmids expressing Myc-tagged full-length AhR or Flag-tagged NR1D1 (Supplementary Fig. 7b). The overexpression of AhR increased the Oil Red O-positive area, whereas co-expression of NR1D1 inhibited the change (Fig. 6j).

TEC-specific AhR knockout attenuated IR- and IS-induced increases in renal FA synthesis, lipid accumulation and fibrosis

Then, we constructed proximal TEC-specific AhR knockout mice (Cre+/AhRfl/fl). Homozygous littermates lacking Ggt1-Cre (Cre−/AhRfl/fl) served as controls (Supplementary Fig. 8a, b). Renal AhR was obviously reduced in Cre+/AhRfl/fl mice compared with Cre−/AhRfl/fl mice (Supplementary Fig. 8c). Immunofluorescence staining further revealed that IR or IS treatment induced an increase in the colocalization of AhR with the proximal TEC-specific marker Megalin and the distal TEC-specific marker sodium-chloride cotransporter (NCC) in Cre−/AhRfl/fl mice. The colocalization of AhR with Megalin disappeared and the colocalization of AhR with NCC persisted in Cre+/AhRfl/fl mice, indicating that AhR deletion was restricted to proximal TECs (Supplementary Fig. 8d, e). No significant differences in renal lipid accumulation and fibrosis was observed between Cre+/AhRfl/fl mice and Cre−/AhRfl/fl mice. Oil Red O staining revealed that IR-induced lipid accumulation was markedly attenuated in Cre+/AhRfl/fl mice compared with Cre−/AhRfl/fl mice (Fig. 7a). The decrease in NR1D1 and increases in SREBP1 and FASN induced by IR were also suppressed in the kidneys of Cre+/AhRfl/fl mice (Fig. 7b, c). In addition, IR-induced increases in renal TG and FFA contents were attenuated in Cre+/AhRfl/fl mice (Fig. 7d). Kidney function tests revealed that the increases in serum IS, creatinine and BUN levels induced by IR were significantly attenuated in Cre+/AhRfl/fl mice (Fig. 7e). HE, Masson and Sirius Red staining revealed that tubular atrophy and collagen deposition induced by IR were attenuated in Cre+/AhRfl/fl mice compared with Cre−/AhRfl/fl mice (Fig. 7f). Increases in FN and α-SMA induced by IR were also attenuated in the kidneys of Cre+/AhRfl/fl mice (Fig. 7g). Similar to the results in IR mice, IS treatment induced renal lipid accumulation, the decrease in NR1D1, and increases in SREBP1, FASN, TG and FFA. Knockout of AhR attenuated the increases in FA synthesis and lipid accumulation (Fig. 8a–d). Moreover, knockout of AhR did not affect serum IS, creatinine or BUN levels (Fig. 8e). Histological analysis and Western blot revealed that IS treatment induced renal damage, collagen deposition, and increases in FN and α-SMA, which were also attenuated by AhR knockout (Fig. 8f, g). These results indicate that knockout of AhR in proximal TECs alleviates the increases in FA synthesis and lipid accumulation, kidney functional deterioration and fibrosis following the accumulation of uremic toxins.

Fig. 7. TEC-specific AhR knockout attenuated the IR-induced increases in renal FA synthesis, lipid accumulation and fibrosis.

Fig. 7

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a Oil Red O staining and quantification of kidneys from sham- or IR-treated Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). Scale bar = 50 μm. b Western blot and quantification showing the protein levels of NR1D1, SREBP1 and FASN in sham- or IR-treated kidneys from Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). c qPCR showing the mRNA levels of Srebf1 and Fasn in sham- or IR-treated kidneys from Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). d Renal TG and FFA contents from sham- or IR-treated Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). e Serum IS, creatinine and BUN levels in sham- or IR-treated Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 5 for IS levels; n = 6 for creatinine or BUN levels). f HE, Masson and Sirius Red staining and quantification of kidneys from sham- or IR-treated Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). Scale bar = 50 μm. g Western blot and quantification showing the protein levels of FN and α-SMA in sham- or IR-treated kidneys from Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). Data were shown as mean ± SD. Statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparisons test (a-g). Source data are provided as a Source Data file.

Fig. 8. TEC-specific AhR knockout attenuated the IS-induced increases in renal FA synthesis, lipid accumulation and fibrosis.

Fig. 8

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a Oil Red O staining and quantification of kidneys from vehicle- or IS-treated Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). Scale bar = 50 μm. b Western blot and quantification showing the protein levels of NR1D1, SREBP1 and FASN in vehicle- or IS-treated kidneys from Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). c qPCR showing the mRNA levels of Srebf1 and Fasn in vehicle- or IS-treated kidneys from Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). d Renal TG and FFA contents from vehicle- or IS-treated Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). e Serum IS, creatinine and BUN levels in vehicle- or IS-treated Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 5 for IS levels; n = 6 for creatinine or BUN levels). f HE, Masson and Sirius Red staining and quantification of kidneys from vehicle- or IS-treated Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). Scale bar = 50 μm. g Western blot and quantification showing the protein levels of FN and α-SMA in vehicle- or IS-treated kidneys from Cre−/AhRfl/fl and Cre+/AhRfl/fl mice (n = 6). Data were shown as mean ± SD. Statistical analysis was performed by two-way ANOVA with Tukey’s multiple comparisons test (a–g). Source data are provided as a Source Data file.

Pharmacological activation of NR1D1 alleviated IR-induced increases in renal FA synthesis, lipid accumulation and fibrosis

NR1D1 agonist SR9009 was injected beginning on the third day after contralateral nephrectomy in IR mice (Fig. 9a). SR9009 did not affect the expression of NR1D1 (Fig. 9b), but the IR-induced decline in kidney function was alleviated by SR9009 (Fig. 9c). Oil Red O staining and morphology analysis revealed that lipid accumulation, tubular atrophy and collagen deposition induced by IR were attenuated by SR9009 (Fig. 9d). The increases in SREBP1, FASN, renal TG and FFA contents, FN and α-SMA induced by IR were also inhibited by SR9009 (Fig. 9e–h).

Fig. 9. Pharmacological activation of NR1D1 alleviated the IR-induced increases in renal FA synthesis, lipid accumulation and fibrosis.

Fig. 9

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a Experimental design of IR-treated mice with SR9009. b Western blot and quantification showing NR1D1 expression in the kidneys from IR-treated mice administered with or without SR9009 (n = 6). c Serum creatinine and BUN levels in IR-treated mice administered with or without SR9009 (n = 6). d Oil Red O, HE and Masson staining and quantification of kidneys from IR-treated mice administered with or without SR9009 (n = 6). Scale bar = 50 μm. e Western blot and quantification showing the protein levels of SREBP1 and FASN in the kidneys from IR-treated mice administered with or without SR9009 (n = 6). f qPCR showing the mRNA levels of Srebf1 and Fasn in the kidneys from IR-treated mice administered with or without SR9009 (n = 6). g Renal TG and FFA contents from IR-treated mice administered with or without SR9009 (n = 6). h Western blot and quantification showing the protein levels of FN and α-SMA in the kidneys from IR-treated mice administered with or without SR9009 (n = 6). Data were shown as mean ± SD. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparisons test (b-h). Source data are provided as a Source Data file.

Discussion

Renal tubular cells, which consume large amounts of ATP for reabsorption and secretion, predominantly rely on FAs as the energy source2. Previous studies have reported that lipid metabolism disturbances are a key feature of CKD21. Both the oxidation and transportation of FAs are impaired in the kidneys of CKD patients and mice22–25. Consistent with previous observations, we observed that the mRNA levels of the genes associated with FA oxidation and transportation were significantly altered in the kidneys of both CKD mice and patients based on analysis of published data. Surprisingly, the mRNA levels of FA synthesis-associated genes were remarkably increased and positively correlated with renal fibrosis markers. Therefore, this study focused on investigating the underlying mechanisms leading to the disturbances in FA synthesis and their relevance in CKD.

Disturbances of FA synthesis have been implicated in organ fibrosis26. SREBP1 and FASN are key regulators of FA synthesis. SREBP1 serves as the primary transcription factor regulating the transcription of FA synthesis-associated genes, and FASN is the rate-limiting enzyme27. The increases in SREBP1 and FASN are closely related to steatosis-induced liver fibrosis and bleomycin-induced pulmonary fibrosis28,29. Inhibition of FASN activity attenuates liver injury, inflammation and fibrosis in patients with nonalcoholic steatohepatitis30. In this study, we observed that the protein and mRNA levels of SREBP1 and FASN were increased in IS-treated mTECs and the kidneys of CKD mice. Furthermore, ¹³C labeling of palmitic acid and stearic acid derived from the glucose tracer was also increased in IS-treated mTECs, indicating that FA synthesis was elevated in renal fibrosis. These results are supported by a previous study in which knockout of Fasn in tubular cells or pharmacological inhibition of FASN protected cultured tubular cells from profibrotic phenotype transition and renal fibrosis31. These results suggested that elevated FA synthesis plays an important role in renal fibrosis.

Some studies have shown that the overload of free FAs may lead to lipotoxicity in the kidneys by disrupting mitochondrial membrane integrity32, triggering inflammation and cell death, and then promoting fibrosis31,33. In this study, we also observed that TG and FFA contents were increased in fibrotic kidneys induced by IR or IS, as well as in IS-treated mTECs.

It is widely accepted that the accumulation of uremic toxins may induce organ damage, including kidney damage7. Previous studies have shown that the accumulation of uremic toxins promotes renal fibrosis13,34. In this study, our analysis revealed that both serum IS and TG were increased in patients with stage 1–5 CKD. IS treatment induced renal fibrosis and increases in FA synthesis and lipid accumulation. These data suggest that the accumulation of uremic toxins may disrupt renal lipid metabolism.

AhR, a receptor for multiple uremic toxins, has been increasingly recognized as a regulator of lipid metabolism. Tischkau SA et al. reported that AhR knockout decreases FA oxidation in the liver of mice35. Xie W et al. reported that persistent AhR activation in mice upregulates hepatic CD36 expression, which enhances FA uptake and ultimately triggers hepatic steatosis36. Consistent with our previous study13, AhR knockdown attenuated the IS-induced deficiency in FA oxidation, as evidenced by changes in OCR, PPARα, CPT1A and ACOX1. These results suggested that AhR promotes lipid accumulation by inhibiting FA oxidation. Furthermore, we also observed that the mRNA levels of AhR were positively correlated with those of FA synthesis-associated genes. Proximal tubule-specific AhR knockout in mice attenuated the increases in FA synthesis, lipid accumulation and renal fibrosis. These results indicated that AhR upregulation and activation are implicated in renal lipid metabolic disturbances by enhancing FA synthesis.

IP‒MS and analysis on the STRING database showed that NR1D1, a protein associated with lipid metabolism, was able to interact with AhR. Molecular docking and Co-IP results revealed that the acidic domain of AhR mediated its interaction with NR1D1, promoting the ubiquitination and proteasomal degradation of NR1D1. Functioning as an adaptor, AhR bridges target substrates to the cullin 4B E3 ubiquitin ligase complex, facilitating their degradation. These effects have been observed for estrogen receptor, androgen receptor9, STING37, and RUNX210. Our results also showed that cullin 4B knockdown alleviated the IS-induced decrease in NR1D1 and increase in NR1D1 ubiquitination.

NR1D1, a distinctive member of the nuclear receptor superfamily20, plays a role in organ fibrosis. For example, the bleomycin-induced decrease in NR1D1 promotes lung fibrosis, and the NR1D1 agonist prevents the progression of lung fibrosis in mice19,38. We observed a pronounced decrease in NR1D1 protein levels in IS-treated tubular cells and in the kidneys of IR- or IS-treated mouse models. NR1D1 agonist SR9009 alleviated the fibrotic phenotype transition in cultured cells and renal fibrosis of mice. We also observed that the decrease in NR1D1 expression was associated with lipid metabolic disturbances in the kidneys. The NR1D1 agonist SR9009 alleviated the increase in renal FA synthesis. By integrating epigenomic analysis and ChIP‒qPCR, we demonstrated that NR1D1 suppressed the transcription of Srebf1 by binding to its promoter region, which is also observed in adipose and liver tissues17,18,39. However, a previous study reported that knockout of NR1D1 alleviated renal injury by suppressing ferroptosis in a folic acid-induced acute kidney injury model40. The reason for this discrepancy needs further investigation.

Still, this study has several potential limitations. (a) The recruitment of tubule-specific Nr1d1 knock-in transgenic mice may provide more compelling evidence to clarify the role of NR1D1 in renal FA synthesis and fibrosis. (b) Our study focused mainly on IS in renal FA synthesis and fibrosis, but it remains unclear whether other uremic toxins will show similar effects. (c) While our study elucidated the downstream mechanisms of AhR, the upstream regulatory pathways of AhR remain unclear.

In summary, we demonstrate that the upregulation and activation of AhR following the accumulation of uremic toxins facilitate the ubiquitination and proteasomal degradation of NR1D1 through its E3 ubiquitin ligase function, which relieves the transcriptional repression on Srebf1. The overactivation of the SREBP1/FASN pathway increases FA synthesis and leads to lipid accumulation and renal fibrosis (Fig. 10). These observations suggest that suppressing lipid overproduction by targeting AhR or NR1D1 might be a potential approach against renal fibrosis.

Fig. 10. The schematic diagram illustrates how AhR regulates renal lipid metabolism and fibrosis.

Fig. 10

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Under pathological conditions of CKD, following the decline in kidney function and accumulation of uremic toxins in body fluids, the uremic toxin receptor AhR is elevated. The upregulation and activation of AhR act as an E3 ubiquitin ligase to facilitate the ubiquitination and proteasomal degradation of NR1D1, which relieves the transcriptional repression of NR1D1 on the SREBP1/FASN pathway. This, in turn, leads to the overproduction of fatty acids, lipid toxicity in renal tubular epithelial cells, and the progression of renal fibrosis. Created in BioRender. Xie, H. (https://BioRender.com/ahry0ch).

Methods

Ethics statement

All human and animal experiments were approved by the Ethics Committee of Shanghai Tongji Hospital (No. K-W-2019-008 and  2023-DW-SB-025, respectively). Written informed consent was obtained from all patients.

Mouse model preparation

Male C57BL/6J and AhR gene knockout mice aged 8–10 weeks were used in this study. An IR-induced renal injury mouse model was established as previously described13. Briefly, the left renal pedicle was clamped for 40 min, and the right kidney was removed on day 28 after surgery. Blood and kidney samples were collected on day 56 after IR surgery.

To establish an IS-induced CKD model, the right kidney was removed 3 days before IS administration via oral gavage (200 mg/kg body weight per day). Blood and kidney samples were collected after 50 days of IS treatment.

For the SR9009 treatment, SR9009 was dissolved in 15% Cremophor EL and 85% sterile water via ultrasound as described previously19. On day 31 after IR surgery, the mice were intraperitoneally administered SR9009 (100 mg/kg body weight per day).

Generation of proximal tubule-specific AhR gene knockout mice

Proximal tubule-specific AhR gene knockout mice were generated by cross-mating of AhR gene-floxed mice (C57BL/6N-Ahrem1.1, Cyagen, Suzhou, China) with Ggt1-Cre mice (012841, The Jackson Laboratory). Primer sequences used for genotyping are provided in Supplementary Table 1.

Cell culture and treatment

Immortalized mTECs were obtained from Immocell (Xiamen, China). Primary TECs were prepared from renal cortices of male Cre+/AhRfl/fl or Cre−/AhRfl/fl mice by digestion with 1 mg/mL collagenase II in phosphate-buffered saline (PBS) at 37 °C for 15 min, followed by sequential filtration through 100 μm and 40 μm cell filters (BD Falcon). The filtrate was centrifuged, and the resulting pellet was resuspended. All cells were cultured in DMEM/F12 medium (HyClone, Logan, Utah) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37 °C in 5% CO₂.

For IS treatment, cells were starved in serum-free DMEM/F12 medium overnight and subsequently treated with IS (1000 μmol/L). Cells were treated with MG132 (10 μmol/L) for 4 h before harvest or with CHX (10 μmol/L) for specified durations. Cells were pretreated with SR9009 (10 μmol/L) for 1 h before IS treatment.

Renal morphological observations

The renal morphology was observed by HE, Masson and Sirius Red staining and scored as previously described41. Briefly, HE staining was used to analyze tubular injury. The degree of tubular injury was quantified by the proportion of renal cortex displaying histopathological features such as brush border loss, tubular dilation and cast formation. Scores were assigned as follows: 0% = 0, <25% = 1, 26–50% = 2, 51–75% = 3, and >75% = 4. Images of kidney sections stained with Masson or Sirius Red were analyzed for interstitial fibrosis by Fiji software according to the proportions of blue and red areas, respectively. Six randomly selected regions of each mouse kidney were analyzed and scored in a blinded manner.

IHC and immunofluorescence staining

Residual human renal biopsy samples were obtained from Shanghai Tongji Hospital. Renal sections were subjected to IHC using an anti-AhR antibody (1:200). Color development was performed using a DAB-containing IHC detection kit.

For immunofluorescence staining, kidney sections and cells were incubated overnight at 4 °C with anti-AhR (1:200) and either anti-Megalin (1:50), anti-NCC (1:200), or anti-E-cadherin (1:100) antibodies, followed by incubation with fluorescein-labeled secondary antibodies (1:200) and mounting with an antifade reagent containing DAPI. The antigens were visualized via confocal microscopy (Leica, Wetzlar, Germany).

Oil Red O staining

Cultured cells and frozen renal sections (10 μm thick) were fixed in 4% paraformaldehyde for 10 min at RT and then immersed in propylene glycol for 5 min. Samples were stained with freshly prepared Oil Red O for 10 min at 60 °C, differentiated in 85% propylene glycol for 1 min at RT, and counterstained with hematoxylin for 1 min. Samples were mounted with glycerol gelatin, imaged by light microscopy (Olympus, Tokyo, Japan) and analyzed with Fiji software.

Biochemical analysis

BUN, creatinine, FFA and TG concentrations were measured by commercially available kits.

The IS concentration was assessed by liquid chromatography/mass spectrometry (LC/MS) as previously described13.

Transfection of siRNA and plasmids

Transfection of siRNA targeting the mouse AhR gene (si-AhR, 5’-UCCCACAUCCGCAUGAUUA-3’) and cullin 4B gene (si-cullin 4B, 5’-GCUGAAUUUAAAGAGGGCAAA-3’), pEnCMV plasmids harboring Flag-tagged mouse Nr1d1 gene (Flag-NR1D1), and pcDNA3.4 plasmids harboring Myc-tagged full-length (AhR WT) and truncated (AhR Δacid and AhR Δα) mouse AhR gene was performed using the Lipofectamine™ 3000 reagent. Cells were infected with an adenovirus containing the Flag-tagged mouse AhR gene or a control adenovirus, as previously described13.

Western blot

Proteins from renal tissues and cells were extracted with 2% sodium dodecyl sulfate (SDS) lysis buffer. Proteins were denatured by boiling, separated via SDS‒PAGE, and then transferred onto a 0.22 μm polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). Membranes were incubated with primary antibodies at 4 °C overnight and then with secondary antibodies. Signals were developed using an enhanced chemiluminescence kit, and target protein levels were quantified relative to GAPDH.

qPCR

RNA from renal tissues and cells was extracted with TRIzol (Biocolor Bioscience, Shanghai, China). cDNA was generated by reverse transcription with the PrimeScript™ RT reagent (RR036A; Takara Bio, Shiga, Japan). The mRNA level was measured by qPCR using TB Green Premix Ex Taq™ reagent (RR420; Takara Bio, Shiga, Japan) and quantified relative to Gapdh. Primer sequences used for qPCR are provided in Supplementary Table 2.

Measurement of OCR

mTECs, treated with IS in the presence or absence of si-AhR, were seeded onto an XFe96 cell culture microplate (Seahorse Bioscience). Before the assay, cells were washed and incubated in pre-warmed Seahorse XF assay medium in a 37 °C incubator without CO₂ for 1 h. Mitochondrial respiration was then assessed using an XF96 Analyzer (Seahorse Bioscience) according to the manufacturer’s protocol for the Mito Stress Test. The assay sequentially injected the following compounds to measure key respiratory parameters: oligomycin (1 µmol/L), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 1 µmol/L), and a mixture of rotenone (0.5 µmol/L) and antimycin A (0.5 µmol/L). All OCR values were normalized to the cell number in each well, as determined by Hoechst 33342 nuclear staining.

Measurement of PLA2 enzyme activity

PLA2 activity was measured using a commercial assay kit according to the manufacturer’s instructions. Briefly, mTECs or kidney tissues were lysed in a low-Triton buffer (20× stock composition: 200 mmol/L Tris pH 7.5, 2 mol/L NaCl, 20 mmol/L EDTA, 0.2% Triton X-100) and then sonicated and homogenized. Following centrifugation, the supernatant was transferred to a 96-well white plate. Fluorescence intensity was monitored at 515 nm using a fluorescence microplate reader (Synergy Neo2, BioTek). The recorded fluorescence values were normalized to either total cell count or kidney tissue weight for further analysis.

Metabolic tracing with [U-¹³C]glucose

mTECs were cultured in DMEM medium supplemented with lipid-free FBS (AB-FBS-DL0500; ABW, Shanghai, China). Then, the cells were serum-starved overnight and subsequently incubated for 48 h in DMEM medium containing [U-¹³C]glucose (2 g/L). Following incubation, cells were washed with PBS, flash-frozen in liquid nitrogen, and subjected to metabolite extraction. The ¹³C enrichment in fatty acids was analyzed using high-performance LC/MS.

Co-IP assay

Cell proteins were extracted using Western blot and IP lysis buffer containing protease inhibitors (Roche, Basel, Switzerland) and phenylmethylsulfonyl fluoride (ST505; Beyotime, Shanghai, China). Total protein (800–1000 μg) was incubated with primary antibodies (3–4 μg) under vertical rotation at 4 °C overnight, followed by incubation with protein A/G-plus agarose beads at 4 °C for 4 h. The beads were washed 5 times with PBS, resuspended in 30 μL 1× SDS loading buffer, boiled at 100 °C for 10 min, and subjected to Western blot.

IP‒MS

mTECs overexpressing Flag‑tagged AhR via adenovirus (Ad-Flag‑AhR) were treated with IS, then collected and subjected to IP using anti-Flag antibodies (n = 1 biological replicate). Immunoprecipitates were separated by SDS‑PAGE and stained with Coomassie blue. Protein bands of interest were excised and digested using trypsin. The resulting peptides were analyzed by LC/MS using an Easy nLC system coupled to a Q‑Exactive HF‑X mass spectrometer (Thermo Scientific). Raw data were searched using MaxQuant (version 2.4.14.0).

ChIP assay

The ChIP assay was performed using a commercial ChIP assay kit following the manufacturer’s instructions and a previous study42. DNA was purified using a DNA purification kit and subjected to PCR or qPCR, with primer sequences provided in Supplementary Table 3.

Antibodies and reagents

An anti-AhR antibody (MA1-514) was purchased from Thermo Scientific (Waltham, MA); an anti-FN antibody (F3648) was purchased from Sigma‒Aldrich (Darmstadt, Germany); anti-NR1D1 antibody (14506-1-AP), anti-FASN antibody (10624-2-AP), anti-α-SMA antibody (14395-1-AP), anti-GAPDH antibody (60004-1-Ig), anti-Flag antibody (20543-1-AP), anti-Myc antibody (16286-1-AP), anti-ACOX1 antibody (68017-1-Ig), anti-CPT1A antibody (15184-1-AP) and anti-ubiquitin (Ub) antibody (10201-2-AP) were purchased from Proteintech (Wuhan, China); anti-SREBP1 antibody (sc-13551) and anti-Megalin antibody (sc-515772) were purchased from Santa Cruz (Dallas, Texas); anti-NCC antibody (ab95302) and anti-cullin 4B antibody (ab76470) were purchased from Abcam (Cambridge, MA); an anti-E-cadherin antibody (610181) was purchased from BD Biosciences (New Jersey); and an anti-PPARα antibody (YM8234) was purchased from Immunoway (Suzhou, China).

IS (I3875) and Oil Red O (O0625) powder were purchased from Sigma‒Aldrich (Darmstadt, Germany). Cremophor EL (S6828) was purchased from Selleck (Houston, TX). SR9009 (HY-16989) and CHX (HY-12320) were purchased from MCE (Shanghai, China). MG132 (S1748), a ChIP assay kit (P2078), a DNA purification kit (D0033), and Western blot and IP lysis buffer (P0013) were purchased from Beyotime (Shanghai, China). A DAB-containing IHC detection kit (GK500710) was purchased from Gene Tech (Shanghai, China). The urea nitrogen assay kit (C013-2-1), creatinine assay kit (C011-2-1) and FFA assay kit (A042-2-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A TG assay kit (E1013) was purchased from APPLYGEN (Beijing, China). An enhanced chemiluminescence kit (180-5001) was purchased from Tanon (Shanghai, China). Protein A/G-plus agarose beads (sc-2003) were purchased from Santa Cruz (Dallas, Texas). A PLA2 enzyme activity kit (E10217) was purchased from Thermo Scientific (Waltham, MA).

Transcriptomic analysis

The renal RNA-seq data of sham 24 h mice and CKD mice subjected to IR for 28 days (GSE98622) were downloaded from the Gene Expression Omnibus (GEO) database. The renal RNA-seq data of healthy individuals and patients with IgA nephropathy (GSE175759) were also downloaded from the GEO database. Samples of healthy individuals (eGFR ≥ 90 ml/min/1.73 m2) were regarded as controls, and samples of IgAN patients with eGFR <90 ml/min/1.73 m2 were regarded as CKD. Heatmaps and correlation plots were drawn with FPKM values (GSE98622) and read count values (GSE175759) of the interest genes. Differentially expressed genes were identified using the limma package (GSE98622). Genes with a fold change > 1.5 and a P value < 0.05 were considered significantly different. GO enrichment analysis of the differentially expressed genes was conducted using clusterProfiler and visualized using ggplot2 in R (version 4.4.0).

Statistics

Statistical analysis was performed using GraphPad Prism 9.0 (La Jolla, CA). Data were shown as mean ± SD. Data distribution was assessed for normality with the Shapiro–Wilk test. When two groups of normally distributed data were compared, a two-tailed unpaired Student’s t-test was used if variances were equal, whereas Welch’s t-test was used if variances were unequal. When more than two groups of data with a normal distribution and one independent variable were compared, the Brown‒Forsythe test was used to assess equal variances, followed by one-way ANOVA with Tukey’s multiple comparisons test. When more than two groups of normally distributed data and two independent variables were compared, two-way ANOVA with Tukey’s multiple comparisons test was used. Spearman’s correlation analysis was used to assess correlations. The sample size (n; number of independent biological replicates) for each statistical analysis was shown in the figure legends. Differences were considered statistically significant when P < 0.05.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information (5.3MB, pdf)
41467_2026_75114_MOESM2_ESM.pdf (179.5KB, pdf)

Description Of Additional Supplementary File

Supplementary Data 1 (69.8KB, xlsx)
Reporting summary (104.3KB, pdf)
Transparent Peer Review file (2.2MB, pdf)

Source data

Source Data (7.1MB, xlsx)

Author contributions

Conceptualization: L.M.L., C.Y., H.X.; Data curation: Y.T.Z., H.X., L.L., X.S., M.N.; Formal analysis: H.X., Y.T.Z., X.X.; Methodology: H.G., Y.N., Y.Y.Z., L.X.L., L.W., J.L., X.W.; Supervision: C.Y., C.X., L.M.L., W.Z., L.Z.; Writing and Review: L.M.L., C.Y., C.X., H.X.

Peer review

Peer review information

Nature Communications thanks the anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Funding

L.M.L. discloses support for the research of this work from the National Natural Science Foundation of China [grant numbers 82570810 and 82370695] and the National Clinical Research Center for Aging and Medicine, Huashan Hospital, Fudan University [grant number 2024KF2004]. C.Y. discloses support for the research and publication of this work from the National Natural Science Foundation of China [grant numbers U25A2032, 82470764 and 82170696]. H.X. discloses support for the research of this work from the National Natural Science Foundation of China [grant number 82300834] and the China Postdoctoral Science Foundation [grant numbers 2023M742648 and 2025T180640].

Data availability

All data in this study are included within the article and its Supplementary information or are available from the corresponding authors upon reasonable request. Mouse and human kidney RNA-seq data used in this study are available in the GEO database under accession codes GSE98622, GSE175759 and GSE137570. The serum metabolite data from healthy controls and patients with stage 1–5 CKD were analyzed from a reported study14. The IP‒MS data for AhR interactome are available within the article and Supplementary Data 1. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Chen Xu, Email: xu_chen85@fudan.edu.cn.

Limin Lu, Email: lulimin@shmu.edu.cn.

Chen Yu, Email: yuchen@tongji.edu.cn.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-026-75114-5.

References

  • 1.Swartling, O., Rydell, H., Stendahl, M., Segelmark, M., Lagerros, Y. T. & Evans, M. CKD progression and mortality among men and women: a nationwide study in Sweden. Am. J. Kidney Dis.78, 190 (2021). [DOI] [PubMed] [Google Scholar]
  • 2.Liu, B. C., Tang, T. T., Lv, L. L. & Lan, H. Y. Renal tubule injury: a driving force toward chronic kidney disease. Kidney Int93, 568–579 (2018). [DOI] [PubMed] [Google Scholar]
  • 3.Lee, L. E., Doke, T., Mukhi, D. & Susztak, K. The key role of altered tubule cell lipid metabolism in kidney disease development. Kidney Int106, 24–34 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Vanholder, R. et al. Review on uremic toxins: classification, concentration, and interindividual variability. Kidney Int63, 1934–1943 (2003). [DOI] [PubMed] [Google Scholar]
  • 5.Schroeder, J. C. et al. The uremic toxin 3-indoxyl sulfate is a potent endogenous agonist for the human aryl hydrocarbon receptor. Biochemistry49, 393–400 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Denison, M. S. & Nagy, S. R. Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu Rev. Pharm. Toxicol.43, 309–334 (2003). [DOI] [PubMed] [Google Scholar]
  • 7.Xie, H., Yang, N., Yu, C. & Lu, L. Uremic toxins mediate kidney diseases: the role of aryl hydrocarbon receptor. Cell Mol. Biol. Lett.29, 38 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Hankinson, O. The aryl hydrocarbon receptor complex. Annu Rev. Pharm. Toxicol.35, 307–340 (1995). [DOI] [PubMed] [Google Scholar]
  • 9.Ohtake, F. et al. Dioxin receptor is a ligand-dependent E3 ubiquitin ligase. Nature446, 562–566 (2007). [DOI] [PubMed] [Google Scholar]
  • 10.Ouyang, L. et al. Indoleamine 2,3-dioxygenase 1 deletion-mediated kynurenine insufficiency in vascular smooth muscle cells exacerbates arterial calcification. Circulation145, 1784–1798 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Cui, W. et al. Gut microbial metabolite facilitates colorectal cancer development via ferroptosis inhibition. Nat. Cell Biol.26, 124–137 (2024). [DOI] [PubMed] [Google Scholar]
  • 12.Arinze N. V. et al. Tryptophan metabolites suppress the Wnt pathway and promote adverse limb events in chronic kidney disease. J. Clin. Invest.132, e142260 (2022). [DOI] [PMC free article] [PubMed]
  • 13.Xie, H. et al. Uremic toxin receptor AhR facilitates renal senescence and fibrosis via suppressing mitochondrial biogenesis. Adv. Sci. (Weinh.)11, e2402066 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Chen, D. Q. et al. Identification of serum metabolites associating with chronic kidney disease progression and anti-fibrotic effect of 5-methoxytryptophan. Nat. Commun.10, 1476 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zuo, S. et al. Lipid synthesis, triggered by PPARgamma T166 dephosphorylation, sustains reparative function of macrophages during tissue repair. Nat. Commun.15, 7269 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Nakamura, H., Nemenoff, R. A., Gronich, J. H. & Bonventre, J. V. Subcellular characteristics of phospholipase A2 activity in the rat kidney. Enhanced cytosolic, mitochondrial, and microsomal phospholipase A2 enzymatic activity after renal ischemia and reperfusion. J. Clin. Invest87, 1810–1818 (1991). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Cho, H. et al. Regulation of circadian behaviour and metabolism by REV-ERB-alpha and REV-ERB-b. eta. Nat.485, 123–127 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Adlanmerini, M. et al. Circadian lipid synthesis in brown fat maintains murine body temperature during chronic cold. Proc. Natl. Acad. Sci. USA116, 18691–18699 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Wang, Q. et al. Circadian clock molecule REV-ERBalpha regulates lung fibrotic progression through collagen stabilization. Nat. Commun.14, 1295 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Zhao, X. et al. Circadian Amplitude Regulation via FBXW7-Targeted REV-ERBalpha Degradation. Cell165, 1644–1657 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Baek, J., He, C., Afshinnia, F., Michailidis, G. & Pennathur, S. Lipidomic approaches to dissect dysregulated lipid metabolism in kidney disease. Nat. Rev. Nephrol.18, 38–55 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kang, H. M. et al. Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development. Nat. Med21, 37–46 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Szeto, H. H., Liu, S., Soong, Y., Alam, N., Prusky, G. T. & Seshan, S. V. Protection of mitochondria prevents high-fat diet-induced glomerulopathy and proximal tubular injury. Kidney Int90, 997–1011 (2016). [DOI] [PubMed] [Google Scholar]
  • 24.Srivastava, S. P. et al. Renal Angptl4 is a key fibrogenic molecule in progressive diabetic kidney disease. Sci. Adv.10, eadn6068 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Yang, X. et al. CD36 in chronic kidney disease: novel insights and therapeutic opportunities. Nat. Rev. Nephrol.13, 769–781 (2017). [DOI] [PubMed] [Google Scholar]
  • 26.Li, J. et al. Tubular ACSM3 deficiency impairs medium-chain fatty acid metabolism and aggravates kidney fibrosis. Proc. Natl. Acad. Sci. USA122, e2505752122 (2025). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Wu, D. et al. Increased mitochondrial fission drives the reprogramming of fatty acid metabolism in hepatocellular carcinoma cells through suppression of Sirtuin 1. Cancer Commun. (Lond.)42, 37–55 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Tang, M. et al. 10-Hydroxy-2-decenoic acid attenuates nonalcoholic fatty liver disease by activating AMPK-alpha signaling pathway. Biochem Pharm.231, 116648 (2025). [DOI] [PubMed] [Google Scholar]
  • 29.Lian, H. et al. Fatty acid synthase inhibition alleviates lung fibrosis via beta-catenin signal in fibroblasts. Life Sci Alliance8, e202402805 (2025). [DOI] [PMC free article] [PubMed]
  • 30.Loomba, R. et al. TVB-2640 (FASN Inhibitor) for the treatment of nonalcoholic steatohepatitis: FASCINATE-1, a randomized, placebo-controlled phase 2a trial. Gastroenterology161, 1475–1486 (2021). [DOI] [PubMed] [Google Scholar]
  • 31.Mukhi, D. et al. ACSS2 gene variants determine kidney disease risk by controlling de novo lipogenesis in kidney tubules. J. Clin. Invest.134, e172963 (2023). [DOI] [PMC free article] [PubMed]
  • 32.Nguyen, T. B. et al. DGAT1-dependent lipid droplet biogenesis protects mitochondrial function during starvation-induced autophagy. Dev. Cell42, 9–21.e25 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Wang, Y. et al. Imbalanced lipid homeostasis caused by membrane alphaKlotho deficiency contributes to the acute kidney injury to chronic kidney disease transition. Kidney Int104, 956–974 (2023). [DOI] [PubMed] [Google Scholar]
  • 34.Sun, C. Y., Chang, S. C. & Wu, M. S. Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation. Kidney Int81, 640–650 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Wang, C., Xu, C. X., Krager, S. L., Bottum, K. M., Liao, D. F. & Tischkau, S. A. Aryl hydrocarbon receptor deficiency enhances insulin sensitivity and reduces PPAR-alpha pathway activity in mice. Environ. Health Perspect.119, 1739–1744 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Lee, J. H. et al. A novel role for the dioxin receptor in fatty acid metabolism and hepatic steatosis. Gastroenterology139, 653–663 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Ma, Z. et al. AhR diminishes the efficacy of chemotherapy via suppressing STING dependent type-I interferon in bladder cancer. Nat. Commun.14, 5415 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Cunningham, P. S. et al. The circadian clock protein REVERBalpha inhibits pulmonary fibrosis development. Proc. Natl. Acad. Sci. USA117, 1139–1147 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Berthier, A. et al. Combinatorial regulation of hepatic cytoplasmic signaling and nuclear transcriptional events by the OGT/REV-ERBalpha complex. Proc. Natl. Acad. Sci. USA115, E11033–E11042 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Guo, L. et al. Targeted inhibition of Rev-erb-alpha/beta limits ferroptosis to ameliorate folic acid-induced acute kidney injury. Br. J. Pharm.178, 328–345 (2021). [DOI] [PubMed] [Google Scholar]
  • 41.Doke, T. et al. NAD(+) precursor supplementation prevents mtRNA/RIG-I-dependent inflammation during kidney injury. Nat. Metab.5, 414–430 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.Mallipattu, S. K. et al. Kruppel-like factor 6 regulates mitochondrial function in the kidney. J. Clin. Invest125, 1347–1361 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Supplementary Information (5.3MB, pdf)
41467_2026_75114_MOESM2_ESM.pdf (179.5KB, pdf)

Description Of Additional Supplementary File

Supplementary Data 1 (69.8KB, xlsx)
Reporting summary (104.3KB, pdf)
Transparent Peer Review file (2.2MB, pdf)
Source Data (7.1MB, xlsx)

Data Availability Statement

All data in this study are included within the article and its Supplementary information or are available from the corresponding authors upon reasonable request. Mouse and human kidney RNA-seq data used in this study are available in the GEO database under accession codes GSE98622, GSE175759 and GSE137570. The serum metabolite data from healthy controls and patients with stage 1–5 CKD were analyzed from a reported study14. The IP‒MS data for AhR interactome are available within the article and Supplementary Data 1. Source data are provided with this paper.

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