PHD锌指蛋白14对肾脏纤维化的抑制效应及机制研究

Kidney fibrosis is the common path for all types of kidney diseases progressing to end stage of renal disease (ESRD). Currently, there is no effective clinical treatments for kidney fibrosis. PHD finger protein (PHF) 14 is a newly identified histone-binding protein. Our previous work showed, severe kidney and lung fibrosis developed at the birth of mice with non-conditional PHF14 knock-out. The expression of PHF14 decreased at the early stage of unilateral ureteral obstruction (UUO) kidney in mice, renal tubule epithelial cells and fibroblast with stimulation of TGF-β in vivo. By the construction of Renal Gene Expression Database (RGED), we revealed the PHF14 expression levels in DNA microarray assay were lower in patients with chronic kidney failure than healthy control and the expression level of PHF14 in kidney biopsy sample in kidney transplant population could predicted the long term survival of graft. Based on the previous data, we plan to construct tamoxifen inducible PHF14 knock-out mice to investigate the effects of PHF14 on kidney fibrosis at the kidney development stage in kidney-targeted PHF14 knock-out mice to avoid the neonatal death caused by respiratory failure, and to discuss the effect of induced PHF14 depletion on kidney fibrosis in adult mouse models. Furthermore, we will verify the role of PHF14 in kidney fibrosis in UUO kidney fibrosis mice, and test the cell phenotypes changes in primary cultured kidney fibroblasts and renal tubule epithelial cells from kidneys of PHF14 knock-out mice, and in kidney cell lines under fibrotic stimulus. Meanwhile, we will test the changes of platelet derived growth factor (PDGF) and Numb related pathway signal proteins in kidney fibrosis inhibition effects of PHF14. After the planned work, we hope to establish the role of PHF14 as an important kidney fibrosis inhibitor and clarify the possible cell signal pathways involved in the kidney fibrosis inhibition driven by PHF14. This study might provide novel strategies for kidney fibrosis treatment and prevention clinically in the future.

肾脏纤维化是所有肾脏损害发展到终末期肾衰竭的共同通路，临床尚无有效治疗手段。PHD锌指蛋白（PHF）14是一种新鉴定的染色质结合蛋白。前期工作显示，PHF14在早期单侧输尿管结扎（UUO）肾纤维化小鼠和肾纤维化患者中表达较低；转染PHF14高表达质粒的肾成纤维细胞，细胞外基质产生降低；而PHF14非条件敲除小鼠出生时即出现显著的肾脏纤维化。在此基础上，拟研究肾脏在各生理阶段和纤维化状态下PHF14的表达分布特征，评价其与纤维化表型的关系；利用体外细胞平台研究PHF14抑制肾纤维化的机制，重点分析与Numb和PDGF信号通路的关联；再进一步构建PHF14诱导敲除和肾组织特异性敲除小鼠模型，探讨在PHF14不同敲除条件下肾纤维化表型和肾功能损害的特点，验证PHF14的抗肾纤维化效应，并探讨可能参与的信号通路。上述研究旨在探索PHF14抑制肾脏纤维化的调控新机制，为临床干预肾纤维化提供理论基础。

肾脏纤维化是所有慢性肾脏病以及急性肾损伤慢性转化病程的共同病理变化，肾纤维化持续进展，最终可导致尿毒症。在缺血、肾毒性药物等急性损害因素打击后，肾脏纤维化的形成机制可由多种机制介导。先前研究新发现了一种在胚胎组织中调节间质生长的因子-PHF14。PHF14敲除的小鼠在出生后若干小时内即死亡，致死原因是包括肾脏在内都多个重要器官严重纤维化。此现象提示PHF14在器官纤维化过程中发挥重要的调节作用，但具体机制尚不明了。本研究的主要目的是阐明在叶酸导致的急性肾损伤模型后续肾纤维化过程中PHF14的表达调节，同时揭示PHF14在肾脏先后病理过程中所发挥的作用。 .本研究发现在小鼠叶酸损害导致的肾纤维化模型中，PHF14表达上调，在离体状态下，TGF-beta刺激大鼠肾成纤维细胞也可以促使PHF14上调，在机制研究中，发现P-smad3作为PHF14表达的转录因子。同样在离体状态下，敲除PHF14会提高由TGF-beta刺激所导致的collagen I和alpha-SMA的合成水平。PHF14参与构成了可以抑制血小板源性生长因子（PDGF）信号通路过度激活的复合物，该复合物发挥PDGF信号抑制作用的机制是选择性抑制血小板源性生长因子受体alpha（PDGFR-alpha）的转录。在体实验，我们使用了phf14 flox/flox； Cre-ER小鼠，在验证了该小鼠诱导敲除phf14基因的效率后，我们证实相比于对照组，phf14敲除组小鼠在经受叶酸诱导急性肾损伤后，肾纤维化的发生更加严重。.上述工作表明，PHF14表达在急性肾损伤后的肾纤维化过程中上调，这一上调是由TGF-beta/smad3信号通路所介导的，上调的PHF14通过抑制PDGFR-alpha的表达阻碍了肾纤维化进程。因此，TGF-beta/smad3/PHF14可被视作在TGF-beta所主导的肾脏促纤维化信号通路中的自限机制，发挥内源性的抗肾脏纤维化作用。