Endothelial to Mesenchymal Transition compromises vascular integrity to induce hypoxia and Myc mediated metabolic reprogramming of tubular epithelial cells in kidney fibrosis

The aim of this study was to identify the functional impact of Endothelial-to-Mesenchymal Transition (EndMT) in kidney fibrosis. For this purpose EndMT has been conditionally deleted in endothelial cells by the genetic deletion of Twist and Snail in the endothelial cells, using the tamoxifen inducible Cdh5-ERT2 Cre model. Twist EndMT–cKO and Snail EndMT–cKO mice, together with WT mice, were challanged with the unilateral ureteral obstruction model (UUO) to induce kidney fibrosis. Mice were euthanized 10 days after surgery. In addition, since we found that EndMT-induced vascular damage and hypoxia induces Myc upregulation in tubular epithelial cells (TECs), we deleted Myc in TECs (using the GGT-Cre model). Myc GGT–cKO mice were also challenged with the UUO model and euthanized 10 days after surgery. Overall design: Transcriptional profile of fibrotic (UUO), healthy or contralateral kidneys from Twist, Snail and Myc conditional knock-out mice (triplicate for each experimental condition) was generated by whole transcriptome RNA-Seq using the Illumina platform.

本研究旨在明确内皮细胞向间充质转化（Endothelial-to-Mesenchymal Transition，EndMT）在肾纤维化中的功能影响。为此，我们采用他莫昔芬诱导型Cdh5-ERT2 Cre模型，在内皮细胞中条件性敲除Twist与Snail基因，以此特异性删除内皮细胞中的EndMT过程。将Twist EndMT条件性敲除（cKO）小鼠、Snail EndMT条件性敲除小鼠与野生型（WT）小鼠均采用单侧输尿管梗阻（Unilateral Ureteral Obstruction，UUO）模型诱导肾纤维化，术后10天处死所有小鼠。此外，本研究发现EndMT介导的血管损伤与缺氧可诱导肾小管上皮细胞（Tubular Epithelial Cells，TECs）中Myc基因上调，因此我们进一步利用GGT-Cre模型在肾小管上皮细胞中条件性敲除Myc基因。将Myc条件性敲除小鼠同样采用UUO模型诱导肾纤维化，并于术后10天处死。总体实验设计：采用Illumina平台进行全转录组RNA测序，获取Twist、Snail及Myc条件性敲除小鼠的梗阻侧纤维化肾脏、健康肾脏及对侧肾脏的转录组表达谱，每组实验条件设置3个生物学重复。