Mesenchymal stromal cells secretome restores bioenergetic and redox homeostasis in human proximal tubule cells after ischemic injury

BACKGROUND: Ischemia/reperfusion injury (IRI) is the leading cause of acute kidney injury (AKI). The current standard of care focuses on supporting kidney function, stating the need for more efficient and targeted therapies to enhance repair. Mesenchymal Stromal Cells (MSCs) and their secretome, either as conditioned medium (CM) or extracellular vesicles (EVs), have emerged as promising options for regenerative therapy, however, their full potential in treating AKI remains unknown.
METHODS: In this study, we employed an in vitro model of chemically-induced ischemia using antimycin A combined with 2-deoxy-D-glucose to induce ischemic injury in proximal tubule epithelial cells. Afterwards, we evaluated the effects of MSC secretome, CM or EVs obtained from adipose tissue, bone marrow and umbilical cord, on ameliorating the detrimental effects of ischemia. To assess the damage and treatment outcomes, we analyzed cell morphology, mitochondrial health parameters (mitochondrial activity, ATP production, mass and membrane potential) and overall cell metabolism by metabolomics.
RESULTS: Our findings show that ischemic injury caused cytoskeletal changes confirmed by disruption of the F-actin network, energetic imbalance as revealed by a 50% decrease in the oxygen consumption rate, increased oxidative stress, mitochondrial dysfunction and reduced cell metabolism. Upon treatment with MSC secretome, the morphological derangements were partly restored and ATP production increased by 40-50%, with umbilical cord-derived EVs being most effective. Furthermore, MSC treatment led to phenotype restoration as indicated by an increase in cell bioenergetics, including increased levels of glycolysis intermediates, as well as an accumulation of antioxidant metabolites.
CONCLUSION: Our in vitro model effectively replicated the in vivo-like morphological and molecular changes observed during ischemic injury. Additionally, treatment with MSC secretome ameliorated proximal tubule damage, highlighting its potential as a viable therapeutic option for targeting AKI.

研究背景（BACKGROUND）：缺血再灌注损伤（Ischemia/reperfusion injury, IRI）是急性肾损伤（acute kidney injury, AKI）的首要致病原因。当前的标准治疗方案以维持肾脏功能为核心，亟需开发更高效、更具靶向性的治疗手段以促进肾脏修复。间充质基质细胞（Mesenchymal Stromal Cells, MSCs）及其分泌组，无论以条件培养基（conditioned medium, CM）还是细胞外囊泡（extracellular vesicles, EVs）形式存在，均已成为再生治疗的潜在候选方案，但其在治疗AKI中的完整潜力仍未明确。
研究方法（METHODS）：本研究采用antimycin A联合2-deoxy-D-glucose构建化学诱导缺血的体外模型，以诱导近端肾小管上皮细胞发生缺血性损伤。随后，我们评估了源自脂肪组织、骨髓及脐带的MSCs分泌组、条件培养基或细胞外囊泡对缺血损伤有害作用的改善效果。为评估损伤程度与治疗结局，我们通过细胞形态学观察、线粒体健康参数（线粒体活性、ATP生成量、质量及膜电位）分析，以及代谢组学检测，全面评估了整体细胞代谢状态。
研究结果（RESULTS）：本研究发现，缺血损伤可引发细胞骨架改变，表现为F-actin网络破坏；同时导致能量失衡，具体体现为耗氧率降低50%，氧化应激水平升高，线粒体功能障碍以及细胞代谢水平下降。经MSCs分泌组处理后，细胞的形态学紊乱得到部分逆转，ATP生成量提升40%~50%，其中脐带来源的EVs治疗效果最为显著。此外，MSCs治疗可促进细胞表型恢复，表现为细胞生物能学指标改善，包括糖酵解中间体水平升高，以及抗氧化代谢物积累。
研究结论（CONCLUSION）：本研究所构建的体外模型可有效模拟缺血损伤过程中体内类似的形态学与分子学变化。此外，MSCs分泌组治疗可改善近端肾小管损伤，表明其有望成为治疗AKI的可行治疗方案。