Cisplatin induces mouse AKI and RTECs pyroptosis.
Cisplatin-induced AKI seriously threatens the health of cancer patients and largely limits its application to chemotherapy [26]. Therefore, it is of great significance to study the pathogenesis of cisplatin-induced AKI. First, we constructed an AKI mouse model by cisplatin induction. We then detected histopathological damage to the renal cortex of cisplatin-induced AKI mice by H&E staining. As shown in Fig. 1A, cisplatin causes a moderate renal tubular injury. The kidney section showed obvious focal tubular epithelial cells swelling, dilation and detachment with moderate tubular vacuole. In addition, the levels of blood urea nitrogen (BUN) (Fig. 1B), serum creatinine (Fig. 1C) and TNF-α (Fig. 1D) were significantly increased in cisplatin-AKI mice model.
Previous study found that pyroptosis plays an important role in many inflammatory diseases, such as cisplatin-induced AKI [23]. Therefore, in this study we further explored how pyroptosis participates in cisplatin-induced AKI. First, we detected the expression of GSDMD in kidney tissue by IHC analysis. As shown in Fig. 1E, the expression of GSDMD in the model group (cp) was significantly increased when compared with the control group. Then, we detected the expression levels of caspase-1 and GSDMD-N by WB analysis. The variation trend of the results is consistent with GSDMD (Fig. 1F-G). These in vivo experiments preliminarily elucidated the involvement of pyroptosis in cisplatin-induced AKI.
In order to further elucidate the role of pyroptosis in cisplatin-induced AKI, we conducted further studies in vitro. HK2 cell lines (human RTECs) were selected for the study. First, the expression of caspase-1 in HK2 cells were detected by IF when treated with cisplatin. As shown in Fig. 1H, the expression level of caspase-1 in HK2 cells treated with cisplatin was significantly increased when compared with the control group. Then, we detected the expression level of GSDMD-N in HK2 cells when treated with cisplatin. The variation trend of the results is consistent with caspase-1 (Fig. 1I-J). In summary, these in vivo and in vitro results indicate that pyroptosis is involved in cisplatin-induced AKI.
HK2 cell-derived exosomes treated with cisplatin influenced pyroptosis of surrounding HK2 cells.
Previous studies have reported that exosomes play a key role in the development of kidney disease. However, there are few reports on cisplatin-induced AKI, and the regulatory mechanism is unclear. Therefore, in the present study, we explored the role of exosomes in cisplatin-induced pyroptosis of HK2 cells. First, HK2 cells were treated with cisplatin and exosome inhibitor GW4869 at the same time, and the expression of caspase-1 was detected by IF. As shown in Fig. 2A, the treatment of HK2 cells with cisplatin can significantly increase the expression of caspase-1, but after adding GW4869, the expression of caspase-1 was significantly down-regulated. This result suggests that exosomes are involved in cisplatin-induced pyroptosis of surrounding HK2 cells. To further verify our hypothesis, we also detected the mRNA and protein expression levels of caspase-1, NLRP3 and GSDMD by qRT-PCR and WB, respectively. The variation trend of the results is consistent with caspase-1 (Fig. 2B-D). Taken together, these results indicate that exosomes are involved in cisplatin-induced AKI pyroptosis.
Next, we focused on the exosomes in HK2 cells. Therefore, we further isolated and identified exosomes from HK2 cells. As shown in Fig. 2E, exosomes were characterized by transmission electron microscope (TEM). TEM analysis of isolated exosomes showed round structures with diameters between 30 and 150 nm. The qNano analysis was used to quantify the particle diameter of the population of small vesicles collected from HK2 cells and the mean diameter of HK2-exo detected was 100 nm (Fig. 2F). We also measured exosome markers CD63 and CD63 in HK2 cells when treated with or without cisplatin. WB assay showed that CD63 and CD63 were all expressed in both ctrl-exo and cp-exo groups (Fig. 2G).
Cisplatin-treated HK2 cells exosome-derived miR-122 regulates pyroptosis in surrounding cells.
In eukaryotes, miRNAs usually regulate gene expression at the post-transcriptional level. Previous studies have found that abnormal levels of miRNA could be one of the mechanisms explaining dysregulated protein expression in the progression of kidney disease [27]. When evaluating an integrative network of miRNAs and mRNA data, miR-122 was found to be one of a possible master regulator in AKI [28]. Therefore, we speculate that miR-122 is involved in the regulation of cisplatin-induced AKI pyroptosis. To verify our hypothesis, we first tested the expression of miR-122 in AKI mice and HK2 cells. As shown in Fig. 3A-B, cisplatin treatment significantly reduced miR-122 expression in vivo and in vitro when compared with control. However, after adding GW4869, the expression of miR-122 significantly increased to close to the control group (Fig. 3C).
To further explore the role of exosomes derived miR-122 in cisplatin-induced pyroptosis of HK2 cells. First, the miR-122 mimic and inhibitor were used to transfect HK2 cells to overexpress (miR-122 OE) and knock down (miR-122 KD) miR-122, respectively. Then, the expression of caspase-1 in surrounding HK2 cells (NC) were detected by IF when adding cisplatin-treated HK2 cell-derived exosomes (cp-exo) and cisplatin-treated miR-122 OE HK2 cell-derived exosomes (cp-exo + miR-122 OE) treatments, separately. As shown in Fig. 3E, the expression level of caspase-1 was significantly increased when compared with the NC group when adding cp-exo treatment. In contrast, the expression level of caspase-1 was significantly decreased when adding cp-exo + miR-122 OE treatment. We also tested the expression of caspase-1 in miR-122KD HK2 cell line. The results showed that the expression of caspase-1 was also significantly increased in miR-122 KD HK2 cell line when compared with the NC group. Next, we also detected the mRNA and protein expression levels of caspase-1, NLRP3 and GSDMD by qRT-PCR and WB, respectively. The variation trend of the results is consistent with caspase-1 (Fig. 3E-G). The above results further indicate that the miR-122 involved in cisplatin-treated HK2 cell exosomes affects the surrounding HK2 cell pyroptosis.
We further explored the regulatory mechanism of miR-122 involved in the regulation of pyroptosis in cisplatin-treated HK2 cells. First, the expression level of miR-122 was detected by qRT-PCR in NC, NC + cp-exo, NC + cp-exo-miR-122 OE and NC + miR-122 KD groups. As shown in Fig. 3H, the expression level of miR-122 was significantly decreased when adding cp-exo treatment. In contrast, the expression level of miR-122 was significantly increased when adding cp-exo + miR-122 OE treatment. We also tested the expression of miR-122 in miR-122KD HK2 cell line. The result was in line with expectations. Then, we predicted the relevant targets. The TargetScan Human 7.2 software was used to reveal that miR-122 targets ELAVL1. As shown in Fig. 3I, ELAVL1 gene was found to contain putative sites of the 3'-UTR untranslated region (3'-UTR) that matched to the miR-122 seed region. To investigate whether miR-122 targets ELAVL1 in cisplatin-induced AKI, we set up the luciferase reporter plasmid (containing the wild-type (WT) and mutation-type (MUT) 3'-UTR) of target gene ELAVL1 by luciferase reporter vector. We further used the transfection reagent to transfect these reporter gene plasmids (WT and MUT) into HK2 cells together with the miR-122 mimic or miR-122 inhibitor, respectively. Compared with control groups, WT reporter activity were predominantly decreased in HK2 cells when transfected with miR-122 mimic. In contrast, WT reporter activity was significantly up-regulated in HK2 transfected with miR-122 inhibitor. While, the transfection of miR-122 mimic and miR-122 inhibitor did not affect the activity of MUT reporter activity (Fig. 3J). Then, we detected the protein expression of ELAVL1 when adding cisplatin treatment in vivo and in vitro. As shown in Fig. 3K-L, the expression level of ELAVL1 was significantly increased when adding cisplatin treatment. To elucidate the role of exosomes in regulating the expression of ELAVL1. We also added GW4869 treatment. The results showed that the expression of ELAVL1 was significantly decreased in HK2 cells when adding cisplatin and GW4869 treatment at the same time (Fig. 3M).
Finally, we conducted a rescue experiment. We measured the protein expression of ELAVL1 in NC, NC + cp-exo, NC + cp-exo-miR-122 OE and NC + miR-122 KD groups. As shown in Fig. 3N, the expression level of ELAVL1 was significantly increased when adding cp-exo treatment. In contrast, the expression level of ELAVL1 was significantly decreased when adding cp-exo + miR-122 OE treatment. We also tested the expression of ELAVL1 in miR-122KD HK2 cell line. The result was in line with expectations. All these results suggest that miR-122 negatively regulates ELAVL1 expression by directly binding to the 3'-UTR region of ELAVL1 in cisplatin-induced AKI.
Exosome-derived miR-122 affects cisplatin-induced AKI and HK2 cells pyroptosis by regulating the expression of ELAVL1.
In order to further explore the role of miR-122-ELAVL1 axis in pyroptosis regulation pathway under cisplatin-induced AKI. First, we transfected the siELAVL1, miR-122 OE + ELAVL1 OE plasmid into HK2 cell line. Then, we extracted the exosomes of these cell lines after cisplatin treatment. Exosomes derived from these cell lines were co-incubated with HK2 cells (NC) to detect the expression of caspase-1 via IF. As shown in Fig. 4A, the expression level of caspase-1 was significantly increased when adding cp-exo treatment. However, the expression level of caspase-1 was significantly decreased when adding cp-exo derived from siELAVL1 HK2 cell line treatment. In contrast, the expression level of caspase-1 was also significantly increased when adding cp-exo derived from miR-122 OE + ELAVL1 OE HK2 cell line treatment. At the same time, we detected the expression of miR-122 and ELAVL1 by qRT-PCR or WB. The experimental results were consistent with the expected results (Fig. 4B-D). Next, we also detected the mRNA and protein expression levels of caspase-1, NLRP3 and GSDMD by qRT-PCR and WB, respectively. As shown in Fig. 4E, the mRNA expression levels of caspase-1, NLRP3 and GSDMD were significantly increased when adding cp-exo treatment. However, the expression levels of caspase-1, NLRP3 and GSDMD were significantly decreased when adding cp-exo derived from siELAVL1 HK2 cell line treatment. In contrast, the expression levels of caspase-1, NLRP3 and GSDMD were also significantly increased when adding cp-exo derived from miR-122 OE + ELAVL1 OE HK2 cell line treatment. The variation trend of caspase-1, NLRP3 and GSDMD protein expression was consistent with that of mRNA (Fig. 4F). Taken together, these results indicate that the miR-122-ELAVL1 axis involved in cisplatin-treated HK2 cell exosomes affects the surrounding HK2 cell pyroptosis.
Finally, we elucidate our hypothesis in vivo. We detected histopathological damage to the renal cortex of cisplatin-induced AKI mice by H&E staining when miR-122 is overexpressed (OE) or ELAVL1 is knocked down (KD). As shown in Fig. 4G, either overexpressing miR-122 or knocking down ELAVL1 will alleviate cisplatin-induced AKI. We also detected the expression of GSDMD in kidney tissue by IHC analysis. As shown in Fig. 4H, the expression of GSDMD in the miR-122 OE and siELAVL1 AKI mice were all significantly decreased when compared with the model group. In addition, the levels of blood urea nitrogen (BUN) (Fig. 4I), serum creatinine (Fig. 4J), TNF-α (Fig. 4K), IL-1β (Fig. 4L) and IL-18 (Fig. 4M) were significantly decreased in the miR-122 OE and siELAVL1 AKI mice. We also tested the expression of miR-122 in the miR-122 OE and siELAVL1 AKI mice (Fig. 4N). At the same time, we detected the expression of ELAVL1, caspase-1, NLRP3 and GSDMD proteins in the miR-122 OE and siELAVL1 AKI mice. The results were in line with expectations (Fig. 4O). All in all, these results indicate that miR-122 inhibits cisplatin-induced AKI and HK2 cells pyroptosis via ELAVL1.