Activation of the YAP/KLF5 transcriptional cascade in renal tubular cells aggravates kidney injury

The Hippo/YAP pathway plays a critical role in early embryonic kidney development, but its functions in the adult kidney are less well understood. Our previous work has demonstrated that tubular YAP activation induced by double knockout of the upstream Hippo kinases Mst1 and Mst2 (Mst1/2 dKO) promotes tubular injury and renal inflammation under basal conditions. However, the importance of tubular YAP activation remains to be established in injured kidneys in which many other injurious pathways are simultaneously activated. Several secreted factors were found to be increased by YAP in tubular cells, but how the transcriptional network is altered by YAP is largely unknown. Here, we show that tubular YAP was already activated at 6 h after unilateral ureteral obstruction (UUO). Tubular YAP deficiency greatly attenuated tubular cell over-proliferation, tubular injury and renal inflammation induced by UUO or cisplatin. RNA-Seq, ChIP and luciferase assay revealed that YAP promoted the transcription of the transcription factor KLF5 whereas no interaction between YAP and KLF5 was observed. Consistently, the elevated expression of KLF5 and its target genes in Mst1/2 dKO or UUO kidneys was blocked by ablation of Yap in tubular cells. Inhibition of KLF5 prevented tubular cell over-proliferation, tubular injury and renal inflammation in Mst1/2 dKO kidneys. Therefore, our results demonstrate that tubular YAP is a key player in kidney injury. YAP and KLF5 form a transcriptional cascade, in which tubular YAP activation induced by kidney injury promotes KLF5 transcription. Activation of this cascade induces tubular cell over-proliferation, tubular injury and renal inflammation Overall design: HK-2 cells cultured in DMEM/F12 containing 1% FBS underwent electrotransfection with GFP or Yap and Taz plasmids. 5 h after electrotransfection, total RNA was extracted. RNA sequencing (RNA-seq) was performed on an Illumina Nova Seq 6000 System (Novogene Co., Ltd.).

Hippo/YAP信号通路（Hippo/YAP pathway）在胚胎早期肾脏发育中发挥关键作用，但其在成年肾脏中的功能尚不完全明确。本团队既往研究证实，上游Hippo激酶Mst1与Mst2双敲除（Mst1/2 dKO）所诱导的肾小管YAP激活，可在基础稳态下促进肾小管损伤与肾脏炎症反应。然而，在多种损伤通路同时激活的受损肾脏中，肾小管YAP激活的生物学意义仍有待明确。已有研究发现YAP可上调肾小管细胞中多种分泌因子的表达，但YAP如何调控转录网络的重塑仍不甚明晰。本研究发现，单侧输尿管梗阻（UUO）造模后6小时，肾小管YAP即已激活。肾小管特异性YAP敲除可显著减轻UUO或顺铂诱导的肾小管细胞过度增殖、肾小管损伤及肾脏炎症。转录组测序（RNA-Seq）、染色质免疫共沉淀（ChIP）及荧光素酶报告基因实验结果显示，YAP可促进转录因子KLF5的转录，且未检测到YAP与KLF5之间存在直接相互作用。与之相符的是，Mst1/2 dKO或UUO模型肾脏中KLF5及其靶基因的表达上调，可被肾小管细胞YAP敲除所阻断。抑制KLF5的活性可阻止Mst1/2 dKO模型肾脏中的肾小管细胞过度增殖、肾小管损伤及肾脏炎症反应。综上，本研究结果证实，肾小管YAP是肾脏损伤进程中的关键调控因子。YAP与KLF5构成一条转录级联通路：肾脏损伤诱导的肾小管YAP激活可促进KLF5的转录，该通路的激活可进一步诱导肾小管细胞过度增殖、肾小管损伤及肾脏炎症反应。整体实验设计：将培养于含1%胎牛血清（FBS）的DMEM/F12培养基中的HK-2细胞，分别以绿色荧光蛋白（GFP）、Yap及Taz质粒进行电转染。电转染5小时后提取总RNA，随后依托Illumina NovaSeq 6000测序系统（诺禾致源生物信息科技有限公司）完成RNA测序（RNA-seq）。