解放军医学杂志2025，Vol.50Issue(12)：1577-1585,9.DOI:10.11855/j.issn.0577-7402.0347.2025.0928
肾脏类器官培养优化及其初步应用效果研究
Optimization of kidney organoid culture and its preliminary application effects
摘要
Abstract
Objective To construct kidney organoids and establish various models of acute and chronic kidney injury.Methods Kidney organoid differentiation was induced from human embryonic stem cells(hESCs)via the CHIR99021-fibroblast growth factor(FGF)activation pathway.Cisplatin-induced acute renal tubular injury models were established by treating kidney organoids with cisplatin at concentrations of 20,30,and 50 μmol/L.The temporal progression of injury was investigated following stimulation with 30 μmol/L cisplatin for 12,24,and 48 h.The expression levels of renal injury markers and inflammatory factors were assessed using reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and immunohistochemistry(IHC).Additionally,a high-glucose-induced fibrosis model with blood glucose fluctuation and a sustained high-glucose model were developed using two approaches:alternating culture media containing 5 mmol/L and 25 mmol/L glucose every other day,and continuous exposure to 25 mmol/L glucose for 6 d.Renal fibrosis markers,collagen Ⅲ(Col Ⅲ),transforming growth factor-β(TGF-β),and fibronectin,were evaluated by Western blotting,RT-qPCR,and IHC.Results Tubular structures began forming in the organoids by day 8,reaching optimal morphology by day 14,suitable for subsequent research.Immunostaining for specific markers,proximal tubules(Lotus tetragonolobus lectin,LTL),distal tubules(cadherin 1,CDH1),and connecting tubules(uromodulin,UMOD),along with hematoxylin and eosin(HE)staining confirmed successful kidney organoid formation.Following cisplatin stimulation,renal injury markers[kidney injury molecule-1(KIM-1),neutrophil gelatinase-associated lipocalin(NGAL)]and inflammatory factors[tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),chemokine(C-C motif)ligand 2(CCL2)]were significantly upregulated,indicating the onset of acute kidney injury.The 30 μmol/L cisplatin treatment induced the most suitable injury model,with NGAL and KIM-1 expression increasing over stimulation time.In both high-glucose models,fibronectin,Col Ⅲ,and TGF-β were markedly elevated,but the glucose fluctuation model was superior to the sustained high-glucose model.Conclusion The optimized culture protocol enables efficient generation of kidney organoids,which can be utilized to establish various acute and chronic kidney injury models.关键词
肾脏/类器官/诱导性多能干细胞/顺铂肾损伤/糖尿病肾病Key words
kidney/organoid/induced pluripotent stem cells/cisplatin-induced kidney injury/diabetic nephropathy分类
医药卫生引用本文复制引用
Shi Wen-Ying,Xu Li-Jia,Rui Hong-Liang,Zhang Sen,Zhou Xun-Rong..肾脏类器官培养优化及其初步应用效果研究[J].解放军医学杂志,2025,50(12):1577-1585,9.基金项目
This work was supported by the Major Collaborative Innovation Project of Beijing(2022-I2M-1-014) 北京市重大协同创新项目(2022-I2M-1-014) （2022-I2M-1-014）
