Circ DENND4C inhibits pyroptosis and alleviates ischemia-reperfusion acute kidney injury by exosomes secreted from human urine-derived stem cells

Acute kidney injury (AKI) is a disease characterised by acute onset, high mortality, and poor prognosis, and is mainly caused by ischemia-reperfusion (I/R). Human urine-derived stem cells (USCs) exhibit antioxidant, antiinflammatory, and anti-apoptotic cytoprotective effects. Previously, we found that exosomes from USCs had the ability to inhibit apoptosis and protect kidneys from I/R injury. This study aimed to investigate the role of USC-derived exosomes (USC-Exos) in reducing pyroptosis and alleviating I/R-AKI. Models of HK-2 cells hypoxiareoxygenation (H/R) and I/R kidney injury was established in Sprague Dawley rats to simulate AKI in vitro and in vivo. USC-Exos were isolated using ultracentrifugation and identified via electron microscopy and western blotting. USC-Exos were co-cultured with HK-2 cells and injected into rats via the tail vein. The expression of pyroptosis-related molecules (GSDMD, caspase-1, and NLRP-3) was verified using PCR and western blotting. Changes in renal function were reflected in the serum creatinine, urea, and cystatin C levels. The degree of renal injury was determined using haematoxylin and eosin and immunohistochemical staining. The levels of IL-1 beta and IL-18 were detected using enzyme-linked immunosorbent assay (ELISA) to verify the role of USC-Exos in pyroptosis. Differentially expressed circRNAs in I/R rat kidneys were screened by transcriptome sequencing, and a dual-luciferase experiment was used to verify the interaction between upstream and downstream molecules. Ischemia-reperfusion resulted in significantly impaired renal function and expression of pyroptosis molecules, and significantly increased concentrations of inflammatory factors. These effects were reversed by injecting USCExos. Circ DENND4C was the most significantly decreased circRNA in I/R rat renal tissue, and knock-down of circ DENND4C can aggravate AKI in vivo and in vitro. DAVID(http://david.abcc.ncifcrf.gov) website showed that miR 138-5p/FOXO3a is a potential downstream target of circ DENND4C. Knock-down of circ DENND4C in HK-2 cells resulted in increased expression of miR 138-5p and increased miR 138-5p can reverse the regulation of FOXO3a. Dual-luciferase assay verified the reverse interaction between circ DENND4C, miR 138-5p, and FOXO3a. Exosomes promote cell proliferation and inhibit the activation of NLR family pyrin domain containing 3 through the circ DENND4C/miR 138-5p/FOXO3a pathway, thereby reducing pyroptosis and AKI. Circ DENND4C may be a potential therapeutic target for AKI.