<i>CircAASS</i> alleviates renal injury and fibrosis by regulating mitochondrial homeostasis in tubular epithelial cells

Acute kidney injury (AKI) is characterized by the dysfunction of renal tubular epithelial cells (TECs), often leading to renal fibrosis. Mitochondrial impairment is a common hallmark across various types of AKI. However, the potential role of circular RNAs (circRNAs) in modulating mitochondrial homeostasis during AKI and subsequent renal fibrosis remains underexplored. Our findings reveal a significant reduction of <i>circAass</i> levels in the renal cortex across all three AKI models. Mechanistically, <i>circAASS</i> mitigates TEC apoptosis and inflammatory responses by promoting mitochondrial homeostasis, thereby attenuating AKI. Specifically, cytoplasmic <i>circAASS</i> acts as a competing endogenous RNA (ceRNA) by sequestering <i>MIR324-3p</i>, which in turn enhances the expression of <i>PINK1</i>, a critical regulator of mitophagy. Additionally, nuclear <i>circAASS</i> directly interacts with the PPARGC1A/PGC-1α protein, inhibiting its ubiquitin-mediated degradation and thereby promoting mitochondrial biogenesis. Furthermore, we demonstrated that the RNA-binding protein IGF2BP2 suppresses <i>circAASS</i> biogenesis by binding to intronic sequences in the <i>AASS</i> pre-mRNA. Restoring <i>circAass</i> in AKI mouse models improves both mitochondrial biogenesis and mitophagy, ameliorating pro-inflammatory responses of TECs and thus mitigating renal fibrosis. Decreased <i>circAASS</i> expression and its association with impaired mitochondrial function in TECs, followed by more severe renal fibrosis, are observed in AKI patients. Collectively, our results suggest that <i>circAASS</i> protects against AKI by regulating mitochondrial homeostasis, highlighting its potential as a therapeutic target for kidney injury.<b>Abbreviations:</b> AAV9: adeno-associated virus serotype 9; AKI: acute kidney injury; BLAST: Basic Local Alignment Search Tool; ceRNA: competing endogenous RNA; circRNA: circular RNA; CKD: chronic kidney disease; CP-AKI: cisplatin-induced AKI; DHE: dihydroethidium; FISH: fluorescence in situ hybridization; HK2: human renal proximal tubular cells; IF: immunofluorescence; H/R: hypoxia-reoxygenation; I/R: ischemia-reperfusion; ISH: in situ hybridization; LPS: lipopolysaccharide; m⁶A: N⁶-methyladenosine; MMP: mitochondrial membrane potential; NC: negative control; ncRNA: non-coding RNA; PAS: periodic acid-schiff staining; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RBPs: RNA-binding proteins; RIP: RNA immunoprecipitation; ROS: reactive oxygen species; RT-qPCR: real-time quantitative polymerase chain reaction; SAKI: septic AKI; Scr: serum creatinine; Seq: sequencing; siRNA: small interfering RNA; TECs: tubular epithelial cells; TEM: transmission electron microscopy.