Exosomal miR-122-5p from tubular cells ameliorates renal interstitial fibrosis by regulating fibroblasts via HIF-1α

Renal interstitial fibrosis (RIF) is a common pathological feature and final manifestation of chronic progressive kidney disease. Although exosome-mediated intercellular communication is known to play a crucial role in RIF, the mechanism by which injured tubular epithelial cells (TECs) contribute to fibrogenesis remains incompletely understood. In this study, we investigated the role of exosomal miR-122-5p derived from TECs in modulating fibroblast activation and renal fibrosis. Exosomes were isolated from kidneys of unilateral ureteral obstruction (UUO) mice and from TGF-β1-stimulated HK-2 cells. These exosomes were either co-cultured with fibroblasts (NRK-49F cells) or injected into UUO mice via the tail vein. High-throughput miRNA profiling was used to identify differentially expressed miRNAs in exosomes derived from HK-2 cells, and miR-122-5p was selected for further investigation based on expression level and bioinformatic prediction. Functional analyses were performed using miRNA mimics, inhibitors, and target validation assays. The results showed that exosomal miR-122-5p was significantly downregulated in both fibrotic kidneys and TGF-β1-induced HK-2 cells. In vivo and in vitro, restoration of miR-122-5p levels markedly attenuated renal fibrosis, as evidenced by the reduction of fibrotic markers including α-smooth muscle actin, fibronectin, and collagen I. Mechanistically, miR-122-5p was found to directly target hypoxia-inducible factor 1-alpha (HIF-1α), thereby inhibiting activation of the TGF-β1/Smad signaling pathway. These findings suggest that decreased expression of miR-122-5p in TEC-derived exosomes promotes renal fibrosis through the HIF-1α/TGF-β1/Smad axis, while reintroduction of miR-122-5p can effectively reverse these effects. Taken together, our study provides new insights into the intercellular communication between TECs and fibroblasts via exosomal miRNAs, and identifies miR-122-5p as a potential therapeutic target for the treatment of renal interstitial fibrosis. In this study, high-throughput sequencing was performed to profile the microRNA (miRNA) expression of exosomes derived from renal tubular epithelial cells (HK-2) with or without TGF-β1 stimulation, and miR-122-5p was identified through literature review and bioinformatics analysis as the focus for further investigation. HK-2 cells were divided into six groups—untreated control, TGF-β1, TGF-β1 plus miR-122-5p mimic, TGF-β1 plus mimic negative control, TGF-β1 plus miR-122-5p inhibitor, and TGF-β1 plus inhibitor negative control—and exosomes were isolated from each group. These exosomes were co-incubated with TGF-β1-pretreated rat fibroblasts (NRK-49F), and the expression of fibrosis markers (α-SMA, collagen I, fibronectin) was assessed by immunofluorescence and Western blot to evaluate fibroblast activation. In vivo, a unilateral ureteral obstruction (UUO) mouse model was established, and exosomes from different HK-2 treatment groups were administered via tail vein injection; renal miR-122-5p levels, serum creatinine, blood urea nitrogen, and histological fibrosis (HE, Masson’s trichrome, Sirius Red staining, and immunofluorescence) were evaluated. Potential miR-122-5p target genes were predicted using PITA, miRanda, and RNAhybrid, identifying HIF-1α as a key candidate, which was validated by wild-type/mutant 3′UTR luciferase reporter assays. Finally, Western blot was used to analyze HIF-1α and TGF-β1/Smad pathway proteins (Smad2, p-Smad2, Smad3, p-Smad3, Smad7, E-cadherin), and HIF-1α overexpression experiments were conducted to confirm that miR-122-5p exerts anti-fibrotic effects by regulating the HIF-1α/TGF-β1-Smad signaling axis.