BACKGROUND: Acute kidney injury (AKI) is a major global public health concern with limited treatment options. While preclinical studies have suggested the potential of mesenchymal stem cells (MSCs) to repair and protect injured kidneys in AKI, clinical trials using transarterial MSCs transplantation have yielded disappointing results. This study aimed to investigate the feasibility and safety of minimally invasive renal subcapsular transplantation of MSCs for treating AKI in a minipig model, ultimately aiming to facilitate the clinical translation of this approach. METHODS: A novel AKI minipig model was established by combining cisplatin with hydration to evaluate the effectiveness of potential therapies. Renal subcapsular catheterization was successfully achieved under ultrasound guidance. Subsequently, the efficacy of renal subcapsular MSCs transplantation was assessed, and the biological role of the tryptophan metabolite kynurenine (Kyn) in AKI was elucidated through both in vivo and in vitro experiments. RESULTS: The method of pre-hydration at 4% of body weight, followed by post-cisplatin (3.8 mg/kg) hydration at 2% of body weight, successfully established a cisplatin-induced AKI minipig model with a survival time exceeding 28 days, closely mimicking the clinical characteristics of typical AKI patients. Additionally, we discovered that multiple MSCs transplantations promoted renal function recovery more effectively than single transplantation via the renal subcapsular catheter. Furthermore, elevated levels of Kyn were observed in kidney during AKI, which activated the aryl hydrocarbon receptor (AhR)-mediated NF-κB/NLRP3/IL-1β signaling pathway in tubular epithelial cells, thereby exacerbating inflammatory injury. CONCLUSIONS: Ultrasound-guided renal subcapsular transplantation of mesenchymal stem cells is a safe and effective therapeutic approach for AKI, with the potential to bring about significant clinical advancements in the future.