Autophagy promotes GSDME-mediated pyroptosis via intrinsic and extrinsic apoptotic pathways in cobalt chloride-induced hypoxia reoxygenation-acute kidney injury.

Cobalt is a transition element that abundantly exists in the environment. Besides direct hypoxia stress, cobalt ions indirectly induce hypoxia-reoxygenation injury (HRI), the main cause of acute kidney injury (AKI), a life-threatening clinical syndrome characterized by the necrosis of the proximal tubular epithelial cells (PTECs) and inflammation. Pyroptosis, a type of inflammatory programmed cell death, might play an essential role in HRI-AKI. However, whether pyroptosis is involved in cobalt chloride (CoCl2)-induced HRI-AKI remains unknown. Autophagy is a cellular biological process maintaining cell homeostasis that is involved in cell damage in AKI, yet the underlying regulatory mechanism of autophagy on pyroptosis has not been fully understood. In this study, the in vitro and in vivo models of CoCl2-induced HRI-AKI were established with HK-2 cell line and C57BL/6J mouse. Pyroptosis-related markers were detected with western blotting and immunofluorescence assays, and results showed that gasdermin E (GSDME)-mediated pyroptosis was involved in the cell damage in HRI-AKI. Specific chemical inhibitors of caspase 3, caspase 8, and caspase 9 significantly inhibited GSDME-mediated pyroptosis, verifying that GSDME-mediated pyroptosis was induced via the activation of caspase 3/8/9. The western blotting and immunofluorescence assays were adopted to detect the accumulation of the autophagosomes, and results suggested that HRI increased the autophagic level. The effects of autophagy on apoptosis and pyroptosis were evaluated using lentivirus transfection assays to knock down autophagy-specific genes atg5 and fip200, and results demonstrated that autophagy induced GSDME-mediated pyroptosis via apoptotic pathways in HRI-AKI. Our results revealed the involvement of GSDME-mediated pyroptosis in CoCl2-induced HRI-AKI and promoted the understanding of the regulatory mechanism of GSDME cleavage. Our study might provide a potential therapeutic target for HRI-AKI, and will be helpful for the risk evaluation of cobalt exposure.

Regulation Networks of Non-Coding RNA-Associated ceRNAs in Cisplatin-Induced Acute Kidney Injury.

Cisplatin is widely used as a chemotherapeutic drug to treat various solid tumors. However, it often induces severe side effects, including nephrotoxicity, which limits its application in clinical settings. Furthermore, the underlying mechanisms of action are unclear. Here, we applied whole-transcriptome RNA sequencing to a cisplatin-induced acute kidney injury (CP-AKI) mouse model to evaluate competing endogenous RNA (ceRNA) networks. We found 4460 mRNAs, 1851 long non-coding RNAs, 101 circular RNAs, and 102 microRNAs significantly differentially expressed between CP-AKI and control mice. We performed gene set enrichment analysis to reveal the biological functions of the mRNAs and constructed non-coding RNA-associated ceRNA networks in CP-AKI mice. Two ceRNA regulatory pathways, Lhx1os-203/mmu-miR-21a-3p/Slc7a13 and circular RNA_3907/mmu-miR-185-3p/Ptprn, were validated using quantitative real-time PCR. The protein-protein interaction network indicated that Il6, Cxcl1, Cxcl2, and Plk1 serve as hub genes and are highly connected with the inflammatory response or DNA damage. Transcription factors, such as Stat3, Cebpb, and Foxm1, regulate gene expression levels in CP-AKI. Our study provides insight into non-coding RNA-associated ceRNA networks and mRNAs in CP-AKI and identifies potential treatment targets.

LncRNA MILIP links YBX1 to translational activation of Snai1 and promotes metastasis in clear cell renal cell carcinoma.

BACKGROUND: Distant metastasis is the major cause of clear cell renal cell carcinoma (ccRCC)-associated mortality. However, molecular mechanisms involved in ccRCC metastasis remain to be fully understood. With the increasing appreciation of the role of long non-coding RNAs (lncRNAs) in cancer development, progression, and treatment resistance, the list of aberrantly expressed lncRNAs contributing to ccRCC pathogenesis is expanding rapidly. METHODS: Bioinformatics analysis was carried out to interrogate publicly available ccRCC datasets. In situ hybridization and qRT-PCR assays were used to test lncRNA expression in human ccRCC tissues and cell lines, respectively. Chromatin immunoprecipitation and luciferase reporter assays were used to examine transcriptional regulation of gene expression. Wound healing as well as transwell migration and invasion assays were employed to monitor ccRCC cell migration and invasion in vitro. ccRCC metastasis was also examined using mouse models in vivo. RNA pulldown and RNA immunoprecipitation were performed to test RNA-protein associations, whereas RNA-RNA interactions were tested using domain-specific chromatin isolation by RNA purification. RESULTS: MILIP expression was upregulated in metastatic compared with primary ccRCC tissues. The increased MILIP expression in metastatic ccRCC cells was driven by the transcription factor AP-2 gamma (TFAP2C). Knockdown of MILIP diminished the potential of ccRCC cell migration and invasion in vitro and reduced the formation of ccRCC metastatic lesions in vivo. The effect of MILIP on ccRCC cells was associated with alterations in the expression of epithelial-to-mesenchymal transition (EMT) hallmark genes. Mechanistically, MILIP formed an RNA-RNA duplex with the snail family transcriptional repressor 1 (Snai1) mRNA and bound to Y-box binding protein 1 (YBX1). This promoted the association between the YBX1 protein and the Snai1 mRNA, leading to increased translation of the latter. Snai1 in turn played an important role in MILIP-driven ccRCC metastasis. CONCLUSIONS: The TFAP2C-responsive lncRNA MILIP drives ccRCC metastasis. Targeting MILIP may thus represent a potential avenue for ccRCC treatment.