探索Wnt/beta-catenin信号通路在蛋白尿肾病中的双相双重作用

Proteinuria, featured in most forms of chronic kidney disease (CKD), carries prognostic significance. Prevailing anti-proteinuric strategies have limited efficacy. Canonical Wnt/beta-catenin signaling is a key pathway mediating kidney injury, repair and fibrosis. Data is lacking on its precise role in protein overload proteinuria. We recently demonstrated that exposure to albumin upregulated and then reduced beta-catenin expression in kidney proximal tubular epithelial cells (PTECs) and that molecular knockdown of beta-catenin in PTECs prompted a pro-apoptotic phenotype during protein overload. Emerging data suggest that beta-catenin protects against acute kidney injury (AKI) and yet suppresses repair by promoting fibrosis in chronic injury. This proposal aims to substantiate such biphasic and dual roles of Wnt/beta-catenin signaling in proteinuric nephropathy...We will first study the impact of protein overload on the expression profile of Wnt ligands/beta-catenin and its transcriptional activity in cultured PTECs, and in a mouse model of protein overload. We will then investigate the potential in vitro anti-apoptotic and antifibrotic effects of overexpressing Wnt/beta-catenin (through molecular knock down of the Wnt antagonist DKK1/ectopic expression of stabilized beta-catenin) in protein-overloaded PTECs. To characterize the putative biphasic, dual roles of Wnt signaling in vivo, we will employ the acute (1 week) and chronic (11 weeks) protein overload models in mice. These time-points have been shown in our pilot experiments to reflect acute and chronic kidney injury in this model, respectively. Overexpression of Wnt/beta-catenin in vivo will be achieved either by using DKK1 antisense oligonucleotide (DKK1-AS) to enhance Wnt or by generating a tamoxifen inducible, tubule-specific beta-catenin conditional activation mouse (tam-beta-catenin cAct). In the acute model, wild-type or tam-beta-catenin cAct mice will be given DKK1-AS or tamoxifen respectively at the time of inducing protein overload, and sacrificed after one week for detection of apoptosis and AKI responses. In the chronic model, WT or tam-beta-catenin cAct mice will be given DKK1-AS or tamoxifen respectively 4 weeks after initiating protein overload, and sacrificed after a further 7 weeks of observation (11 weeks of protein overload totally) for analysis of renal fibrosis...Our results will: (i) elucidate the expression profile of Wnt/beta-catenin proteins in PTECs and in animals during acute and chronic protein overload; (ii) provide a definitive answer on whether Wnt/beta-catenin plays biphasic and dual roles of protecting against AKI and yet promoting fibrosis during chronic injury; (iii) stimulate further research into manipulating Wnt/beta-catenin signaling during the different stages of kidney injury to conceive optimal anti-proteinuric strategies.

Wnt/beta-catenin信号通路是肾脏损伤修复和纤维化的关键通路，但其在蛋白尿肾病中的作用仍未阐明。我们发现蛋白超载可引起近端肾小管上皮细胞（PTECs）的beta-catenin表达出现先上升再下降的变化。beta-catenin可能对急性肾损伤有保护作用，而在慢性肾损伤中却促进纤维化的形成而影响修复。我们将首先在体外模型中，研究蛋白超载对PTECs的Wnt/beta-catenin信号通路表达模式及转录活性的影响以及激活此信号通路对蛋白超载引起的PTECs凋亡及纤维化作用。其次将通过条件激活小鼠特异性激活PTECs的Wnt/beta-catenin信号通路，研究其在蛋白超载诱导的急慢性肾损伤动物模型中的双相双重作用。本研究将首次全面阐明蛋白尿肾病发生发展过程中Wnt/beta-catenin信号通路的作用机制，有可能为临床治疗蛋白尿肾病提供崭新的策略。

蛋白尿是慢性肾病的重要特征及预后指标，目前仍无有效治疗手段。经典Wnt/β-catenin信号通路是参与肾脏病理生理损伤的关键信号通路，但是其在蛋白尿肾病中的作用仍未阐明。本研究于体外培养人近端小管上皮细胞系HK-2细胞，并使用人血白蛋白（human serum albumin, HSA）刺激HK-2细胞构建体外蛋白超载模型，借助基因沉默等实验技术手段深入探讨了Wnt/β-catenin信号通路在HSA刺激下HK-2细胞内的活性，及其对HK-2细胞凋亡的影响和作用机制；同时，本研究还借助Cre-loxP系统首次成功构建了近端肾小管上皮细胞（proximal tubule epithelial cell, PTEC）β-catenin条件性基因激活小鼠，即Tubcat小鼠（Ksp-CreERT2-Catnblox(ex3)/wt, Tubcat mouse），并以此转基因小鼠和野生型小鼠为基础，通过构建单侧肾切除术联合腹腔注射小牛血清白蛋白诱导的蛋白尿肾病小鼠模型，深入探讨了PTEC内持续活化的Wnt/β-catenin信号通路对蛋白尿肾病肾组织结构和功能的影响及其作用机制。我们的研究结果显示：（1）Wnt/β-catenin信号通路在蛋白尿肾病肾皮质内和HSA刺激下的HK-2细胞内呈现动态改变。在蛋白尿肾病和HSA刺激的早期，肾皮质和HK-2细胞内的Wnt/β-catenin信号通路出现显著激活，活化的Wnt/β-catenin信号通路能够促进肾脏炎症反应从而加重蛋白尿肾病肾脏结构和功能损害；在蛋白尿肾病和HSA刺激的晚期，肾皮质和HK-2细胞内Wnt/β-catenin信号通路出现显著受抑，失活的Wnt/β-catenin信号通路能够加重PTEC的凋亡并促进蛋白尿肾病肾脏结构和功能损害；（2）在蛋白尿肾病和HSA刺激的晚期，Dickkopf-3（Dkk-3）在肾皮质和HK-2细胞内显著升高，使用重组Dkk-3刺激HK-2细胞可以抑制Wnt/β-catenin信号通路的活性。本项目通过分子、细胞和动物模型三个层次的交叉渗透研究深入阐明了经典Wnt/β-catenin信号通路在蛋白尿肾病不同时期中的作用和机制，为寻找蛋白尿肾病新型治疗方法提供了理论依据。