Introduction

Acute kidney injury (AKI) is a common clinical syndrome and a major health issue, which refers to a rapid decline of kidney function in a short period of time caused by a variety of factors, such as renal hypoperfusion, trauma, sepsis, and toxic drugs, etc [1,2,3]. Approximately 13.3 million people suffer from AKI every year [4], and 30 ~ 70% of AKI patients could develop into chronic kidney disease (CKD) or end-stage kidney disease (ESKD) [5], and about 1.7 million of the deaths are caused by AKI [6, 7]. As we known, cisplatin is an antitumor chemotherapy drug, but one-third of cancer patients receiving cisplatin chemotherapy are susceptible to AKI [8]. However, practical strategies for treating cisplatin-induced AKI are still lacking. Therefore, focusing on the mechanism of cisplatin-induced AKI is of great significance for drug discovery and the improvement in the quality of the population associated with cisplatin nephrotoxicity.

The increasing studies reported that cisplatin could stimulate oxidative stress and induce cellular senescence [7h). These results indicated that the inhibition of H3K27me3 on AhR gene promoters was weakened, and the expression of AhR was up-regulated in cisplatin-induced AKI mice.

Effect of EZH2 inhibition on AhR-mediated tubular epithelial cell senescence

As previously mentioned, AhR regulated cellular senescence and affected EZH2 expression in injured kidneys. However, whether EZH2 participates in the process of AhR-mediated senescence is unknown. To solve the problem, we assessed anti-senescent effect of EZH2 inhibitor zld1039 with AhR agonist FICZ in TCMK-1 cells [24, 27]. Cisplatin triggered the upregulation of SAGs (p16, p21, p53), and EZH2 inhibitor zld1039 repressed senescent mRNA level (Fig. 8a). Furthermore, whether AhR affected senescence through the direct or indirect regulation of EZH2 remain unclear. Here, we used to FICZ agonist activate AhR with the inhibition of EZH2 by zld1039 in TCMK-cells. Consequently, the senescent mRNA level of p16, p21 and p53 were increased following AhR agonist, while EZH2 inhibitor suppressed their corresponding expression (Fig. 8b). These results indicated that the upregulation of EZH2 is necessary for AhR to accelerate cisplatin-induced senescence.

Fig. 8: Effects of EZH2 inhibition on AhR agonism-mediated tubular epithelial TCMK-1 cell senescence.
figure 8

a The mRNA level of senescence related p16, p21, and p53 in cisplatin-induced AKI with or without EZH2 inhibitor zld1039 (EZH2i, n = 4). b The mRNA level of senescence related p16, p21 and p53 in AhR activation (AhRa) by FICZ with zld1039 (n = 4). Data are represented as means ± SDs. cis, cisplatin. ****P < 0.0001, ***P < 0.001, **p < 0.01, *p < 0.05.

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Discussion

Illustrating the potential mechanism of AhR-mediated cellular senescence is of great significance in identifying the cisplatin-induced AKI therapeutic targets. In this study, a novel finding was proposed that AhR-associated cellular senescence was involved in cisplatin-induced AKI. We found that the abnormal expression of AhR was positively associated with cisplatin-induced senescence. Inhibition of AhR by BAY2416964 or tubule-specific gene deletion significantly suppressed cellular senescence and alleviated cisplatin-induced kidney injury. Notably, further studies indicated that EZH2, a histone methyltransferase, was a pivotal factor in AhR-mediated cellular senescence in kidney injury.

Cellular senescence is a proven process of AKI maladaptive repair and kidney fibrosis [10]. A previous study has demonstrated that repeated low-dose cisplatin led to cell cycle arrest at the G2/M phase and cellular senescence, which indicated cellular senescence played a role in cisplatin-induced kidney injury [28]. Here, an increase of β-galactosidase activity, a known characteristic of senescent cells, SAGs (p16, p21, p53) and SASPs (IL-1β, IL-6, TNF-α) were also observed in the kidneys of cisplatin group compared with that of control mice. These results are consistent with those of Li et al. who confirmed that cisplatin-induced cellular senescence in tubular epithelium, could accelerate the progression of renal fibrosis [9].

AhR is an essential ligand-activated transcription factor [29]. A distinct time-dependent and tissue-specific AhR activation is displayed in different mouse models of kidney diseases [30]. Growing evidence presented that AhR activation by uremic toxins, like indoxyl sulfate, plays a harmful role in CKD progression [31, 32]. Whereas, the role of AhR in AKI remains controversial [17, 18]. Tao et al. thought that AhR activation alleviated renal injury in rhabdomyolysis and IR-induced mice by inhibiting inflammation and apoptosis [17]. Eleftheriadis et al. demonstrated that AhR pathway activation enhanced DNA damage response and promoted primary proximal renal tubular epithelial cells senescence, eventually leading to IR-induced kidney injury [18]. Therefore, the contradictory effect of AhR was explored in our study. Here, our results revealed that AhR activation accelerated the progression of kidney injury through a cellular senescence-related mechanism in a cisplatin-induced AKI mice. The AhR inhibition by BAY2416964 or tubule-specific gene deletion repressed cisplatin-induced cellular senescence, which implied that AhR might be one of the causative mechanisms of cisplatin-associated cellular senescence, and inhibition of AhR may be a promising therapeutic strategy against AKI.

Although the relationship between AhR and tubular senescence was observed in our study, we cannot clear the possible mechanisms of AhR-mediated tubular senescence in kidneys of cisplatin-induced mice. EZH2, a catalytic subunit of polycomb repressive complex 2 (PRC2), is an H3K27 histone methyltransferase [33]. Another point noticed is that H3K27me3 is mainly responsible for silencing genes, so it usually acts as a transcriptional suppressor [34]. It has been reported that EZH2 played a significant role in multiple tumor progression by affecting cellular senescence [35, 36]. In particular matter-induced skin keratinocytes, skin senescence depended on AhR-induced ROS as well as the decrease in EZH2 and H3K27me3 [37]. Meanwhile, AhR activation could enhance the EZH2 activity and increase its epigenetic silencing activity, which is a risk factor for environmental toxicant-associated pancreatitis and pancreatic cancer [26]. Not only that, EZH2 binds to the AhR promoter to repress the expression of AhR gene [38]. From this, we thought that EZH2 might play a role in AhR-mediated cellular senescence. Herein, we firstly investigated the correlation between AhR and EZH2. As expected, our results revealed that the mRNA level of AhR and EZH2 were significantly correlated in kidney RNA-sequencing data of cisplatin-stimulated mice. Cisplatin stimulation concurrently up-regulated AhR and EZH2 expression, and AhR inhibitor BAY2416964 or tubule specific AhR deficiency suppressed the expression of EZH2. In vitro, the expression of AhR and EZH2 were increased in cisplatin-treated TCMK-1 cells. The AhR knockdown reversed the elevation of AhR as well as EZH2, suggesting that EZH2 may be a potential mediator in AhR-mediated cellular senescence. Furthermore, we found that EZH2 inhibitor zld1039 indeed improved cisplatin-induced cellular senescence. Activating AhR by agonist FICZ and inhibiting EZH2 by zld1039 repressed the mRNA level of senescent p16, p21 and p53 in cisplatin-stimulated TCMK-1 cells, suggesting that EZH2 is necessary for AhR to accelerate cisplatin-induced cellular senescence.

Importantly, considering the crucial role of EZH2 in epigenetic regulation and combining with the previous results of Ko et al. [38], we further explored the influence of EZH2 knockdown on the expression of AhR. Surprisingly, EZH2 silencing reversely repressed the upregulation of AhR in cisplatin-treated TCMK-1 cells. To illustrate how EZH2 regulates AhR, we used a ChIP assay to examine the enrichment between EZH2 or H3K27me3 and the AhR promoter regions. Consequently, H3K27me3 is responsible for exerting transcriptional inhibition effect in kidneys of control mice, because of the rich enrichment between H3K27me3 and AhR promoter region, which repressed the expression of AhR. Nevertheless, the weaken enrichment of them has been described in kidneys of cisplatin mice, indicating that the rare enrichment of H3K27me3 and AhR promoter might cause cisplatin-induced AhR expression. These results identify that EZH2 also is one of the positive regulators of AhR expression by affecting the enrichment between H3K27me3 and AhR promoter.

In conclusion, our finding demonstrated that AhR was abnormally expressed in kidneys of cisplatin-induced mice and AhR inhibition alleviated cisplatin-induced cellular senescence and tubular injury against AKI. Mechanistically, our study indicated that AhR and EZH2 have mutual regulation, which accelerated tubular senescence in cisplatin-induced AKI. Notably, the crucial role of AhR is the potential to become a promising target for AKI.

Material and methods

Agents and antibodies

Cisplatin and BAY 2416964 were purchased from Synguider (Chengdu, China) and Selleck Chemicals (America), zld1039 was presented by the State Key of Laboratory of Biotherapy, Sichuan University (Sichuan, China), and FICZ was purchased from Selleck Chemicals (America), respectively. All primary antibodies were displayed in Supplementary Table 1.

Animal experiments

Animal experimental procedures were licensed and permitted by the Animal Care and Use Ethics Committee of Sichuan University (2020205 A). Male C57BL/6 J mice were clarified previously [24]. C57BL/6 J mice were administered cisplatin (20 mg/kg) with or without BAY 2416964 (20 mg/kg) by intraperitoneal injection. Tubule specific AhR knockout mice were obtained from the GemPharmatech Co.,Ltd. (Jiangsu, China). AhR tecKO mice were injected cisplatin intraperitoneally to induce AKI.

Cell culture and treatment

Mouse renal tubular epithelial cells (TCMK-1) were purchased from ATCC agency Shanghai Limai Biological Engineering Co., Ltd (Shanghai, China) and were cultured in MEM medium (G4550-500ML, Sercicebio) containing 10% fetal bovine serum (FBS) (SH30084.03, Hyclone) at 37 °C under 5% CO2-95% air environment. TCMK-1 cells were starved in 0.5% FBS medium for 6 h and then treated with 10 μg/ml cisplatin for another 24 h. The siRNAs were used to knock down AhR and EZH2. The transfection procedure is detailed in the riboFECT™ CP transfection kit instruction. AhR-siRNA, EZH2-siRNA and negative control (NC) siRNA were designed and synthesized by GenePharma (Shanghai, China). The detailed transfection sequences information of them is provided in Supplementary Table 2.

Public single-cell RNA-seq analysis

The single-cell RNA sequencing database from http://humphreyslab.com/SingleCell/ was used for single-cell data analysis. According to the database, we checked the single-cell RNA sequencing data about AhR in the healthy adult humans, healthy mice, and ischemia-reperfusion injury mouse kidneys.

Renal function

An automatic biochemical analyzer (Mindray BS-240) was taken to assess Scr and BUN. We defined that the cisplatin-induced AKI mouse model was successfully established, when the Scr value in the cisplatin group was higher than twice that in the control group.

Pathological examination

The kidney tissues were fixed, embedded, and sectioned for Periodic Acid-Schiff (PAS) staining. Renal tubular damage semi-quantitatively scores were used to assess the pathological injury. The specific score standards and detailed rules have been shown in previous study [24].

Immunofluorescence staining

Paraffin kidney tissue sections were firstly deparaffinized and dehydrated. Then, using the microwave method repairs the antigen. After antigen retrieval, the sections were sealed with 1× horse serum containing 0.3% Triton (Sigma, America) for 1 h at 37 °C. And then, they were incubated with primary antibody (the concentration is determined according to the instructions) overnight at 4 °C. Washing the sections and incubating the corresponding secondary antibody and lectin (1:400 dilution) for 1 h at room temperature. Then, the sections were rewashed. DAPI (D8200; Solarbio) was used to stain nuclei for 5 min. Finally, 50% glycerin was used to seal the sections. Photographs were collected from ZEN 2012 microscopy software [39].

Senescence β-galactosidase staining

The senescence β-galactosidase staining kit (Cell Signaling Technology) was used to detect β-galactosidase activity, a known characteristic of senescent cells. For SA β-Gal staining of frozen renal tissues, frozen sections were fixed with 1× fixative solution for 10–15 min at room temperature. Washing the sections with 1×PBS. Then, added the β-galactosidase staining solution to the sections and incubated them at 37 °C overnight in a dry incubator (no CO2). The senescent cells showed blue color under a microscope.

ChIP assay

Proteins and DNA interaction was evaluated by ChIP-qPCR using the ChIP assay kit (Millipore, MA, USA). The experiment protocols were according to the manufacturer’s instructions. The antibodies used for the ChIP assay were as follows: anti-H3k27me3 (Cell Signaling Technology) and control IgG (Millipore). The primers used for ChIP were as follows: AhR-F 5’-GTCAACGACATTTGCGTCCT-3’, AhR-R 5’-TCCCCTTAAGAATTTCAACTGTCC-3’. The calculation formula for enrichment efficiency was elaborated on previously [24].

Western blot analysis

Western blotting was carried out as described earlier [24]. Densitometry analysis was evaluated by using ImageJ 6.0 software (National Institutes of Health, Bethesda, MD, USA). Gray density was normalized using internal reference proteins GAPDH or Histone 3. To ensure the repeatability of the experiment, all immunoblot bands were repeated three times.

Quantitative real-time PCR

Total RNA separation and purification steps and RT-qPCR protocols were displayed as previously shown [24]. The corresponding gene primers were listed in Table S2. The 2−ΔΔCt method was used to calculate the relative gene quantities, and GAPDH was used as the internal reference gene.

Statistical analysis

All quantitative data were presented as mean ± standard deviation (SD). Statistical difference comparisons between the two groups were performed using the T-test. Comparisons between three or multiple groups were performed using a one-way analysis of variance (Tukey’s test). Prism 9.0 (GraphPad Software, San Diego, CA, USA) was used to draw statistical graphs, and P-value less than 0.05 was considered statistically significant.