Development of a Fetal Kidney Failure Model through Nephron Progenitor Cell Ablation via the Intrinsic Apoptosis Pathway

Background: Fetal organ failure models are promising for xenotransplantation targeting fetuses and developmental and pathological research. Conventional knockout models often result in fetal lethality, while tamoxifen-inducible conditional ablation models are harmful to fetuses and lack both rapidity and completeness. In this study, we introduce inducible caspase 9 (iC9) to mouse fetuses for the first time, creating a viable CKD model with an easily adjustable phenotype. Methods: We developed Six2-iC9 mice that express iC9 in nephron progenitor cells (NPCs). To generate a congenital CKD model, we administered a chemical inducer of dimerization (CID) to pregnant mothers intraperitoneally, introducing NPC ablation in Six2-iC9+/+ offspring. This system's efficiency and speed of cell ablation were verified in the culture of Six2-iC9+/+ and Six2-iC9+/- fetal kidneys. Additionally, we developed loxP-iC9 mice and Six2-iC9 rats. Results: A single intraperitoneal CID administration resulted in complete NPC ablation, with the severity adjustable depending on timing. Offspring treated at E13.5 survived, exhibiting significant increases in BUN and urinary albumin from as early as one month old. Histology demonstrated reduced glomeruli with compensatory hypertrophy, tubular dilation, and interstitial fibrosis. In whole-organ cultures of Six2-iC9+/+ fetal kidneys, NPC apoptosis began 6 hours after CID addition, with an ablation efficiency of 94%. Ablation was not induced in Six2-iC9+/-, confirming the existence of a threshold for iC9 expression. However, in dissociated and re-aggregated sphere cultures of Six2-iC9+/- kidneys, NPC ablation was achieved. RNA-seq suggested that DNA damage from cell dissociation made cells more susceptible to apoptosis. Even in whole-organ cultures, co-administering XIAP inhibitor with CID facilitated NPC ablation in Six2-iC9+/-. NPC ablation was also induced in loxP-iC9 mice crossed with Six2-Cre mice and in Six2-iC9 rats. Conclusion: We established a congenital CKD model utilizing the intrinsic apoptosis pathway. Our findings indicate that iC9 expression levels and cell conditions influence this system's effectiveness. The successful implementation of the loxP system promises applications in other cell types, and the development of rat models will advance fetal xenotransplantation research. Funding: Government Support – Non-U.S.