Cisplatin induces acute kidney injury and pyroptosis in mice through the exosome miR-122/ELAVL1 regulatory axis

Although cisplatin is an effective chemotherapeutic drug for the treatment of various cancers, its clinical application is limited due to its side effects, especially nephrotoxicity. Unfortunately, Acute kidney injury (AKI) caused by cisplatin remains one of the main obstacles to cancer treatment. Increasing evidence suggests that renal inammation and pyroptotic inammatory cell death of tubular epithelial cells (RTECs) mainly determine the progression and outcome of cisplatin-induced AKI. However, it is not clear how cisplatin regulates the pyroptosis of RTECs cells in AKI. The current study aimed to determine the regulation mechanism of AKI induced by cisplatin. In vivo, cisplatin was used to induce AKI. H&E staining of mouse kidney tissue sections and serological indicators of kidney injury (including BUN, serum creatinine and TNF-α) were detected. The important substrate protein GSDMD and key target caspase-1 of pyroptosis were detected by immunohistochemistry and western blot, respectively. In vitro, cisplatin signicantly induces HK-2 cells pyroptosis. Furthermore, Cisplatin transmits HK2 cells pyroptosis signals to surrounding cells in the form of exosomes. Previous studies have shown that exosomes are involved in kidney physiology and the pathogenesis of various kidney diseases/disorders. MicroRNAs (miRNAs) are the main functional components of exosomes. exosome miR-122 negatively regulates the pyrolysis of HK2 cells. Finally, we elucidated that exosome miR-122 participates in cisplatin induced AKI regulation by regulating ELAVL1 expression. ELAVL1 under cisplatin


Introduction
Cisplatin is a heavy metal compound with a widely clinical application in various tumors. It is one of the most extensive, effective and valuable adjuvant chemotherapy drugs for treating diverse malignant tumors, including ovarian cancer, cervical cancer and so on [1]. Regardless of its potential medicinal effects, nephrotoxicity caused by cisplatin is one of the main factors limiting its clinical application [2].
Approximately one-third of patients treated with cisplatin suffered from renal dysfunction and injury, especially acute kidney injury (AKI), which has become the most di cult problem in the application of cisplatin [3]. Unfortunately, there is currently no effective treatment to prevent cisplatin-induced AKI [4].
Therefore, it is of great signi cance to explore the regulation mechanism of cisplatin-induced AKI.
AKI is characterized by a sudden decrease in renal function, manifested by an increase in serum creatinine ≥ 0.3 mg/dL or a decrease in urine output of 0.5 mL/kg/h, and is closely related to high morbidity and mortality [5]. The mechanisms of cisplatin-induced AKI are complex, mainly manifested as excessive in ammation and programmed cell death of renal tubular epithelial cells (RTECs) [6]. In the kidney, RTECs are the main target of drug-induced nephrotoxicity and are particularly vulnerable to cisplatin injury [7]. Previous studies have also shown that the common histological feature of cisplatininduced AKI is renal damage caused by renal tubular cell death [8].
Pyroptosis is a new type of proin ammatory programmed cell death regulated by caspases discovered and con rmed in recent years. Its morphological feature is the formation of plasma membrane pores, leading to the secretion of intracellular components and in ammatory cytokines [9]. Early research rst observed the phenomenon of pyroptosis in several phagocytes, such as macrophages [10]. However, recent studies have found that pyroptosis can also be triggered in other types of cells, such as RTECs [11]. A growing number of studies have focused on the role of pyroptosis in AKI. Caspase-11-mediated pyroptosis of RTECs promotes acute renal injury induced by septic [12]. The TNF-α/HMGB1 in ammation signal axis participates in the progression of AKI disease by regulating pyroptosis [13].
Acetylbritannilactone can alleviate contrast-induced AKI by reducing pyroptosis [14]. Furthermore, increasing evidences indicate that RTECs pyroptosis and renal in ammation are involved in the regulation of cisplatin-induced AKI progression and outcome. For example, the cleavage of GSDMD by caspase-11 may promotes cisplatin-induced AKI by triggering RTECs pyroptosis [15]. These studies indicate that the pyroptosis of RTECs is a key event in the occurrence and outcome of cisplatin-induced AKI. However, the exact mechanism by which cisplatin induces AKI by promoting pyroptosis is unclear.
Exosomes participate in cell-to-cell communication with recipient cells by fusing with receptor cell membranes and delivering biomolecules such as proteins, DNA, mRNAs and miRNAs [17]. In recent years, more and more studies have revealed the function of exosomes in the kidney. On the one hand, exosomes can be used as a marker for early diagnosis and treatment of kidney disease [18]; On the other hand, exosomes target effector cells through the paracrine pathway to play a role in kidney pathophysiology [19]. Previous study found that injured renal tubular epithelial cells activate broblasts through exosome miRNAs to promote renal brosis [20]. Limb remote ischemic preconditioning induces systemic upregulation of exo-miR-21, exerting anti-in ammatory and anti-apoptotic effects, thereby protecting sepsisinduced AKI [21]. However, the regulatory mechanism of exosomal miRNAs in cisplatin induced AKI is unclear.
To investigate whether exosomal miRNAs plays a role in cisplatin-induced AKI by regulating pyroptosis, we rst used cisplatin to induce AKI mouse model. Then, we analyzed the expression of GSDMD, a key substrate protein for pyroptosis, in AKI mice by immunohistochemistry (IHC). We also detected the expression of pyroptosis-related proteins caspase-1 and GSDMD-N by Western blot (WB) analysis. The results showed that pyroptosis occurred when cisplatin induced AKI in mice. Next, we studied in vitro. We treated HK2 cell line with cisplatin and detected the expression of caspase-1 by immuno uorescence (IF).
We also measured the expression of GSDMD-N in HK2 cell line by WB. The experimental results are consistent with animal levels. Therefore, we further studied the regulatory mechanism of cisplatin induced AKI. When we added cisplatin and GW4869 at the same time, the expression of caspase-1, NLRP3 and GSDMD was signi cantly down-regulated. These results indicate that cisplatin regulates HK2 cell pyroptosis through exosomes. Further in vitro and in vivo, the gain-or loss-of function experiments were done to clarify that cisplatin affects pyroptosis of RTECs through the exosomal miR-122/ELAVL1 regulatory axis in cisplatin induced AKI.

Materials And Methods
Cell lines and culture conditions HK2 cells (human renal tubular epithelial cells) were purchased from the Chinese Academy of Sciences, Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). These cells were grown in DMEM containing 5% FBS (37 °C, 5% CO 2 ). The HK2 cells were treated with or without 20 µM cisplatin for 24 h.

Animal model of AKI
Adult male C57BL/6 mice (8-12 weeks of age) were obtained from Model Animal Research Center of Nanjing University, Nanjing, China. All animal procedures have been approved by the Institutional Animal Experimental Ethics Committee. 20 mg/kg cisplatin was injected. Cisplatin-induced AKI mice model was constructed as described previously [22]. These mice were intraperitoneally injected with 20 mg/kg body weight cisplatin, and equal volume of 0.9% saline were injected as controls. After 72 hours, the mice were killed under anesthesia. We collected blood samples and kidney tissues for further study.

Serum Creatinine, BUN, TNF-α and Histology
We used a Beckman creatinine analyzer II (DXC800; Beckman Coulter) to analyze the serum creatinine concentration in model mice. BUN was measured using a BUN Assay Kit (Nanjing, China) according to the manufacturer's instructions. The TNF-α levels were detected using the TNF-α Assay Kit (Nanjing, China) according to the manufacturer's instructions. A 2 µm-thick section was used for hematoxylin-eosin staining (H&E) to assess renal tubular histological damage. Immunohistochemistry Para n-embedded renal tissue sections in AKI mice were used to detect the expression of GSDMD (ab219800; 1:1000; Abcam, UK), a marker of pyroptosis, by immunohistochemistry (IHC). The speci c experimental procedures of IHC refer to previous reports [23].

Immuno uorescence staining
The HK2 cells were placed on glass slides in 6-well plates. After cisplatin treatment for 24 h, the cells were xed in 4% paraformaldehyde, blocked with 2% BSA and incubated with 0.1% Triton X-100. The HK2 cells were incubated with caspase-1 antibody (sc-398715, Santa Cruz Biotechnology, 1:200, USA) at 4 °C overnight. The slides were subsequently incubated with secondary antibody (Alexa Fluor 488-labelled). The nuclei were stained using DAPI. The speci c experimental procedures of IHC refer to previous reports [24].

RNA extraction and qRT-PCR
The extraction of total RNA and the analysis of qRT-PCR were performed according to the previous description [1]. We used TRIZOL reagent (Thermo sher, USA) to extract total RNA by in cells and tissues. Taqman probes (Applied Biosystems, USA) were used to quantify miRNAs. Brie y, 1 µg of total RNA was transcribed to cDNA using AMV reverse transcriptase (Takara, Japan) and a RT primer. The reaction conditions were: 16 °C for 30 min, 42 °C for 30 min and 85 °C for 5 min. Real-time PCR was performed using a Taqman PCR kit on an Applied Biosystems 7300 sequence detection system (Applied Biosystems, USA). The reactions were performed in a 96-well plate at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 sec and 60 °C for 1 min. U6 was used as the internal control. Primers: Caspase-1 FP

Exosome isolation and labeling
Exosome-depleted FBS was used in the following experiments to avoid the impact of exosomes. FBS was depleted of exosomes by ultracentrifugation at 1 × 10 6 g at 4 °C for 16 h (Beckman Coulter Avanti J-30I, USA). After being incubated for 48-72 h, the culture medium was harvested and exosomes were isolated by ultracentrifugation. Brie y, cell culture medium was sequentially centrifuged at 300 g for 10 min, 2,000 g for 15 min, and 12,000 g for 30 min to remove oating cells and cellular debris. These were then passed through a 0.22-µm lter. The supernatant was further ultracentrifuged at 1 × 10 6 g for 2 h at 4 °C, washed in phosphate-buffered saline (PBS), and submitted to a second ultracentrifugation in the same conditions. Exosomes were quanti ed with bicinchoninic acid (BCA) method. Exosomal protein was measured by BCA protein assay kit (Beyotime Biotechnology, Nantong, China). The nal exosome pellets were used immediately.

Transmission electron microscope
Exosomes were precipitated and immediately xed in 2.5% glutaraldehyde at 4 °C for the electron microscope observation. After xation, specimens were processed through dehydration in gradient alcohol, and in ltrated in epoxy resin and then embedded. The ultrathin sections were stained with uranyl acetate and lead citrate, and were observed under transmission electron microscope (TEM) (JEM-1010; JEOL, Tokyo, Japan). NanoSight tracking analysis (NTA) Isolated exosomes were analyzed using Nanosight LM10 system (Nanosight Ltd, Navato, CA) equipped with a blue laser (405 nm). Nanoparticles were illuminated by the laser and their movement under Brownian motion was captured for 60 seconds and the video recorded was subjected to NTA using the Nanosight particle tracking software to calculate nanoparticle concentrations and size distribution.
Luciferase reporter assay pMIR-ELAVL1-3'-UTR-WT as well as pMIR-ELAVL1-3'-UTR-MUT luciferase reporter plasmids were constructed by Synthgene Biotech (Nanjing, China). The sequences that could binding to miR-122 were partly mutated and inserted into the reporter plasmid in order to identify the binding speci city. The implementation method refers to previous study [25]. Brie y, HK2 cells were seeded in a 24 well plate until reaching 60% con uence. Each well was co-transfected with luciferase reporter plasmids (0.5 µg) and RNA mimics (100 pmol) using Lipofectamine 2000 (Thermo sher, USA) according to the manufacturer's protocol. The luciferase activity was measured after 48 h of transfection, by using the Dual-Luciferase Reporter Assay (Promega, Shanghai, China) according to the manufacturer's instructions and normalized to Renilla signals. siRNA preparation and transfection ELAVL1 knockdown was accomplished by transfecting cells with siRNA. ELAVL1 and control siRNA were synthesized by Synthgene (China). The speci c method refers to the previous description [25]. The sequences were as follows: the ELAVL1 sense GAACATGACCCAGGATGAGTT, the ELAVL1 antisense UUUUGAAGAAGAAUCGUUGcc. SiRNA transfection into HK2 cells was carried out using Lipofectamine 2000 (Thermo sher, USA) according to the manufacture's instruction.

Overexpression plasmid construction
Full-length ELAVL1 from the human cDNA library was cloned into a pcDNA3.1 vector (Invitrogen; Thermo Fisher Scienti c, Inc.). The pcDNA3.1 vector alone (empty plasmid) served as a negative control. HK2 cells were transfected with pcDNA3.1/ ELAVL1 vector (100 nM) or pcDNA3.1 (100 nM) using Lipofectamine 2000 (Thermo sher, USA) according to the manufacture's instruction. Following transfection for 48 h, the cells were collected for subsequent experiments. The sequence of the ELAVL1 overexpressed plasmid is detailed in the supplementary materials.

Statistical analyses
All experiments were repeated three times and the data are presented as the mean ± standard deviation using SPSS 18.0 (SPSS, inc.). One-way ANOVA and post hoc Dunnett's T3 test were performed in order to compare the differences among and between groups, respectively. P < 0.05 was considered to indicate a statistically signifcant result.

Results
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 signi cance 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 signi cantly increased in cisplatin-AKI mice model.
Previous study found that pyroptosis plays an important role in many in ammatory 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 signi cantly 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 signi cantly 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 in uenced 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 signi cantly increase the expression of caspase-1, but after adding GW4869, the expression of caspase-1 was signi cantly down-regulated. This result suggests that exosomes are involved in cisplatininduced 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 identi ed 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 rst tested the expression of miR-122 in AKI mice and HK2 cells. As shown in Fig. 3A-B, cisplatin treatment signi cantly reduced miR-122 expression in vivo and in vitro when compared with control. However, after adding GW4869, the expression of miR-122 signi cantly 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 signi cantly increased when compared with the NC group when adding cp-exo treatment. In contrast, the expression level of caspase-1 was signi cantly 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 signi cantly 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 + cpexo, NC + cp-exo-miR-122 OE and NC + miR-122 KD groups. As shown in Fig. 3H, the expression level of miR-122 was signi cantly decreased when adding cp-exo treatment. In contrast, the expression level of miR-122 was signi cantly 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 wildtype (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 signi cantly 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 signi cantly 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 signi cantly 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 signi cantly increased when adding cp-exo treatment. In contrast, the expression level of ELAVL1 was signi cantly 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 signi cantly increased when adding cp-exo treatment. However, the expression level of caspase-1 was signi cantly decreased when adding cp-exo derived from siELAVL1 HK2 cell line treatment. In contrast, the expression level of caspase-1 was also signi cantly 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 signi cantly increased when adding cp-exo treatment. However, the expression levels of caspase-1, NLRP3 and GSDMD were signi cantly 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 signi cantly 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 signi cantly 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 signi cantly 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.

Discussion
Cisplatin is an effective chemotherapy drug and has been widely used to treat solid tumors. However, it usually causes AKI in these patients. Previous studies have found that renal insu ciency can independently predict a poor prognosis for cancer patients [29]. However, to date, the mechanism of cisplatin-induced AKI has not been studied in depth, and there is still no effective treatment. The pathological features of AKI are renal tubular cell damage, in ammation and vascular dysfunction. Among them, renal tubular cell death is a common histopathological feature of AKI, and it is generally considered to be one of the factors leading to impaired renal function [30]. Pyroptosis is a proin ammatory programmed cell death regulated by caspases, and its role in kidney damage has been gradually discovered in recent years [9,31]. The role of pyroptosis of RTECs in cisplatin-induced AKI has been reported [12]. In this study, we also used cisplatin induction to construct AKI mouse models, and detected the expression of GSDMD, caspase-1 and GSDMD-N, the key targets of pyroptosis by IHC and WB, respectively (Fig. 1A-G). Then the HK2 cell line was used for in vitro experiments. The results show that cisplatin treatment of HK2 cells can signi cantly increase the expression of caspase-1 and GSDMD-N through IF and WB, respectively (Fig. 1H-J). These results suggest that pyroptosis is involved in cisplatin-induced AKI regulation. This is consistent with previous reports. However, the regulatory mechanism is not yet clear. Therefore, studying the molecular mechanism of RTECs pyroptosis in cisplatin-induced AKI has become an exploration area for the development of new AKI therapies.
Exosomes act as an important medium of communication between cells. Exosomes participate in immune regulation, information transmission and antigen presentation [32]. Substances encapsulated in exosomes, especially miRNAs, play a vital role in the pathogenesis of various kidney diseases [33].
Exosomes derived from hBMSCs can prevent renal ischemia/reperfusion (I/R) injury through miR-199a-3p transplantation [34]. Exosome miRNA-19b-3p of RTECs promotes the activation of M1 macrophages during kidney injury [35]. However, there are few studies on exosome miRNAs in cisplatin-induced AKI, and the mechanism is not clear. In the present study, when we rst added cisplatin and GW4869 to treat HK2 cells at the same time, we found that the phenomenon of pyroptosis was suppressed ( Fig. 2A-D), which initially explained our hypothesis. Therefore, we further isolated and identi ed the exosomes of HK2 cells (Fig. 2E-G). Previous studies found that LncRNA XIST regulates the expression of ASF1A/BRWD1M/PFKFB2 through sponge adsorption of miR-212 and thus participates in the regulation of acute renal injury in renal transplantation [36]. In the present study, we measured the expression of miR-122 in AKI mice and HK2 cells when adding cisplatin treatment. As shown in Fig. 3A-B, when cisplatin was added, the expression of miR-122 was signi cantly down regulated. We further added GW4869 treatment and found that the expression of miR-122 was signi cantly restored (Fig. 3C). To verify the role of miR-122 in cisplatin-induced pyroptosis of HK2 cells. We constructed miR-122 OE and miR-122 KD cell lines. Subsequently, we added cisplatin treatment to the miR-122 OE cell line and extracted the exosomes, and incubated the exosomes with the HK2 cell line. We then tested the relevant indicators of pyroptosis. We also measured the relevant indicators of pyroptosis in miR-122 KD HK2 cell line. The results showed that miR-122 can inhibit the effect of cisplatin-treated HK2 cell exosomes on the pyroptosis of surrounding HK2 cells (Fig. 3D-G).
Next, we identi ed ELAVL1 as a speci c target for miR-122 to regulate pyroptosis in HK2 cells (Fig. 3I). Previous studies found that MALAT1 regulates renal tubular epithelial pyroptosis by modulated miR-23c targeting of ELAVL1 in diabetic nephropathy [37]. However, the role of ELAVL1 in cisplatin-induced AKI has not been reported. In the present study, we rst clari ed the relationship between miR-122 and ELAVL1 through the luciferase assay (Fig. 3J). Then, we detected ELAVL1 protein expression in cisplatintreated mice and cells. The results showed that cisplatin treatment signi cantly increased the expression of ELAVL1 (Fig. 3K-L). Finally, we clari ed that cisplatin-treated HK2 cell exosomes affect the surrounding HK2 pyroptosis (Fig. 3M-N). Finally, we clari ed miR-122 inhibits cisplatin-induced AKI and HK2 cells pyroptosis via ELAVL1 through a functional back ll experiment (Fig. 4).
In general, to our understanding, this study rst described that miR-122 affects the pyroptosis of RTECs in cisplatin-induced AKI by regulating the expression of ELAVL1. This provides a new perspective for the treatment of AKI.

Declarations
Ethics approval and consent to participate Not applicable.

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Availability of data and materials
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Competing interests
The authors declared that there are no con icts of interest.

Funding
Funding information is not applicable.
Authors' contributions BZ and XSY designed and analyzed the experiments and were major contributors to the manuscript.The remaining authors conducted separate experiments, and all of them read and approved the nal manuscript.