The high expression of renal tubular epithelial AhR aggravates cisplatin-induced AKI
The characteristics of AhR in kidneys by public data of single-cell sequencing were shown in Supplementary Fig. 1. Notably, there was an increase in the AhR expression across proximal tubule (PT) clusters after renal ischemia reperfusion injury at 4h (21). In this regard, we could propose that the expression of AhR in PT cells is up-regulated in IR-induced transient renal injury. Thus, based on the above single-cell data, we turn to consult the Gene Expression Omnibus database (No. GSE106993) and checked the RNA-sequencing data about cisplatin-stimulated mice (22). A clustered heatmap was shown in Fig. 1a, the RNA level of AhR was significantly upregulated in the kidneys of cisplatin-induced mice compared with that of control. Subsequently, we used immunofluorescence staining to detect the location and expression of AhR protein in cisplatin renal toxicity mice. In the kidneys of cisplatin mice, the fluorescence intensity of AhR was enhanced in the renal tubule nucleus, indicating the upregulation and activation of AhR in tubules (Fig. 1b). The expression of AhR by an antagonist BAY2416964 treatment was markedly reduced. Consistently, the mRNA and protein levels of AhR were increased after cisplatin stimulation, whereas both of them were substantially decreased by BAY2416964 treatment (Fig. 1c, d).
Importantly, the inhibition of AhR with BAY2416964 reduced the Scr, BUN levels, and kidney injury biomarker Kim-1, NGAL mRNA expression after cisplatin injection (Figure 1e). While the corresponding indicators remained unchanged when treated with BAY2416964 alone (Figure 1e). Meanwhile, the PAS staining displayed that AhR repression improved kidney pathological damages in the cisplatin-induced AKI mice, which was characterized by renal tubular dilation, loss of brush border, cast formation, and tubular epithelial cell apoptosis or necrosis (Figure 1f).
Consistent with the above results, the role of AhR was explored in tubular epithelial cell-specific deletion (tecKO) mice. The upregulation and activation of AhR were observed in WT cisplatin mice, but not in tecKO cisplatin mice (Figure 2a-c). Moreover, the kidney function as well as the mRNA and protein expression of Kim-1, NGAL were dramatically elevated in cisplatin-injected WT mice. As expected, conditional knockout of AhR played a positive renal protective effect, and the increase of corresponding indicators was not found in cisplatin-induced tecKO mice (Figure 2d-f, h). Importantly, conditional knockout of AhR could alleviate kidney pathological damages in cisplatin-induced AKI (Figure 2g). Taken together, these data indicated that the inhibition of AhR protected against cisplatin-induced kidney injury.
AhR promotes cellular senescence in cisplatin-induced AKI mice
Generally, the injured kidney could become a normal or near-normal kidney through the following tissue repair mechanisms, such as inflammatory infiltrate resolution, tubular proliferation, and epithelial repair or regeneration (23). However, maladaptive repair causes cell cycle arrest in the G2/M phase and releases senescence-associated secretory phenotypes (SASPs), which are the most critical risk factor of CKD progression after AKI (23). To explore whether AhR-induced kidney injury is related to cellular senescence, we detected the expression of senescence-associated β-galactosidase (SA β-gal) in the kidneys of cisplatin-induced AKI mice and the SA β-gal activity was significantly increased in the cisplatin-induced renal tubular cells, which was inhibited by AhR suppression with BAY2416964 (Figure 3a). Meanwhile, we also observed an obvious upregulation of senescence-associated genes (SAGs: p16, p21 and p53) and SASPs (IL-1β, IL-6, TNF-α) in the cisplatin mice by RT-qPCR and WB, and further BAY2416964 treatment repressed the expression of the corresponding SAGs and SASPs (Figure 3b-e). It has been reported that AhR activation in the heart generates excessive ROS(24), which may be responsible for cellular senescence (25). Thus, oxidative stress i was evaluated in the kidney of cisplatin-induced AKI mice. Consistent with previous results (26), xanthine oxidase (XO) level was increased, and anti-oxidant enzymes (Cat, Sod1, Sod2) levels were decreased in the cisplatin mice (Figure 3f). These results support a conclusion in which AhR activation induces oxidative stress by stimulating oxidase and repressing antioxidant enzymes. All above, AhR activation in cisplatin-induced AKI mice may show cellular senescence phenotype, which may be mediated by oxidative stress, whereas the specific mechanisms between them needs to be further explored.
Consistently, in cisplatin-induced AhR tecKO mice, lower SA β-gal expression was detected compared with WT mice (Figure 4a). Furthermore, the expression of SAGs (p16, p21 and p53) and SASPs (IL-1β, IL-6, TNF-α) were substantially inhibited in AhR specific deficiency cisplatin mice (Figure 4b-e). Similarly, the oxidase up-regulation and antioxidant enzymes down-regulation in WT cisplatin mice were observed, which were not present in AhR tecKO cisplatin mice. Combined with the above results are sufficient to confirm that AhR activation participates in regulating oxidative stress and cellular senescence in injured kidneys.
Knockdown of AhR alleviates cellular senescence and oxidative stress in cisplatin-stimulated TCMK-1 cells
Next, we carried out an AhR-siRNA transfection test to see if AhR knockdown could exert anti-cellular senescence and anti-oxidant stress effect in vitro. The best silencing efficacy AhR-siRNA#1 from the three transfection sequences was selected, and the mRNA and protein levels of AhR-siRNA#1 were markedly reduced (Figure 5a, b). After cisplatin stimulation, the mRNA levels of Kim-1 and NGAL were significantly up-regulated, while, AhR knockdown improved cisplatin-induced cell injury (Figure 5c). Consistent with the in vivo result, in cisplatin-induced TCMK-1 cells, the downregulation of AhR attenuated cisplatin-stimulated cellular senescence (Figure 5d, e), as evidenced by the reduction of SAGs (p16, p21 and p53) and SASPs (IL-6, TNF-α). In terms of oxidative stress, compared with negative control group, cisplatin stimulation up-regulated XO and down-regulated Sod1, Cat, while AhR knockdown reversed the expression of oxidative stress-related factors and suppressed oxidative stress response (Figure 5f-h).
AhR regulated EZH2 expression in cisplatin-injured kidneys and TCMK-1 cells
Previously, we have demonstrated that inhibition of EZH2 could reduce cisplatin-induced inflammation and improve renal injury (27). In pancreatic cancer cells, the activation of AhR/EZH2 signaling axis causes epigenetic alteration (28). Therefore, we explored the effect of AhR on the regulation of EZH2 in the kidneys of cisplatin-induced AKI mice. We made a correlation heatmap of our previous RNA-sequencing data and confirmed that the mRNA levels of AhR was highly related to EZH2 (Figure 6a). As expected, the expression of EZH2 and a transcription inhibition histone mark, H3K27me3 (Figure 6b, c), together with that of AhR (Figure 1b-d), were dramatically elevated in the kidneys of cisplatin-induced mice. Notably, both the transcription and translation levels of EZH2 were synchronously inhibited by AhR inhibition with BAY2416964 (Figure 6b, c), indicating that the expression of EZH2 is potentially regulated by AhR. Next, we used AhR tecKO mice to further verify the relationship between AhR and EZH2. Similarly, the upregulation of EZH2 and H3K27me3 were reversed by AhR deficiency after cisplatin stimulation (Figure 6d, e). In addition, as elaborated in Figure 5a, b and Figure 6f, g, AhR knockdown repressed the mRNA and protein expression of AhR and EZH2, simultaneously. Moreover, the expression of AhR and EZH2 were enhanced after cisplatin stimulation in vitro, whereas this upregulation was substantially reversed by AhR knockdown (Figure 6h, i). These results powerfully demonstrate that AhR upregulates the expression of EZH2, which may be positively correlated with the progression of tubular cell senescence in cisplatin-induced AKI mice.
AhR and EZH2 may exert physiological effects through reciprocally regulation
As found in the above experimental results, AhR regulates the expression of EZH2. However, as an essential epigenetic regulatory enzyme, EZH2 may play a transcriptional regulation role by affecting H3K27me3. Therefore, to explore whether EZH2 reversely regulates AhR, we further used EZH2 siRNA to detect the expression of AhR in vitro. The best silencing efficiency EZH2-siRNA#1 to transfect TCMK-1 cells was selected, and the mRNA and protein levels of EZH2 were dramatically reduced (Figure 7a, b). If AhR only regulates the expression of EZH2 unidirectionally, EZH2 knockdown does not affect AhR expression level. Actually, the expression of AhR was synchronously inhibited in TCMK-1 cells by EZH2 knockdown (Figure 7c, d), indicating that EZH2 silencing could repress the expression of AhR. Moreover, the expression of EZH2 and AhR in cisplatin-treated TCMK-1 cells was found to be markedly higher than that in the untreated group, while their expression no longer rose by EZH2 silencing (Figure 7e-g). These data further highlight the potential effect that EZH2 regulates AhR expression, reversely.
Nevertheless, how does EZH2 affect the expression of AhR? Whether it has a relationship with the epigenetic effect of EZH2 or H3K27me3? In order to address this question, we used a ChIP assay to detect the enrichment between EZH2/H3K27me3 and AhR gene promoters in vivo. There are significant overlap peaks between AhR and EZH2 promoters in non-renal cells based on the ChIP-Atlas database. Hence, we designed AhR promoter primers on the basis of the overlapping peaks, and used the ChIP-qPCR assay to detect the enrichment between EZH2 and AhR gene promoters. No enrichment between EZH2 and AhR gene promoter regions was found (data not shown). Additionally, AhR-binding peaks significantly overlapped with H3K27me3 in renal cells on the genome level. ChIP-qPCR assay confirmed that H3K27me3 was bound to the AhR promoter regions in the control mice, while cisplatin stimulation reduced this enrichment (Figure 7h). These results indicate that the inhibition of H3K27me3 on AhR gene promoters was weakened, and the expression of AhR was up-regulated in cisplatin-induced AKI mice.