TGF-beta Pathways that Protect Epithelia in Chronic Renal Injury

 慢性肾脏病（CKD）是一个日益严重的公共卫生问题，影响10%的美国人，增加发病率和死亡率，并造成巨大的经济负担。持续性肾小管损伤是肾小管间质纤维化（TIF）的重要组成部分，TIF是任何病因的进行性CKD的共同特征。生长因子如转化生长因子β（TGF β）是肾小管上皮细胞对损伤反应的重要决定因素.尽管过度的TGF-β信号传导促进TIF，但使用遗传工具阻断TGF-β导致CKD小鼠模型后肾小管损伤增加，表明TGF-β信号传导也具有保护作用。TGF-β 1介导许多细胞凋亡效应，包括细胞周期进程的G1期阻滞和上皮细胞去分化。本研究旨在验证近曲小管中TGF β 1信号通过诱导细胞周期停滞和上皮细胞去分化促进慢性肾损伤后存活的假说。第一个人工智能研究如何TGF-β 1改变细胞周期的进展，以及这些变化如何影响上皮细胞的生存，使用小鼠模型的损伤和药理学诱导剂的G1期阻滞。为了测试这一点，我们将使用遗传修饰的小鼠，缺乏TGF β受体（T β RII），特别是在近端小管和诱导慢性肾损伤的血管紧张素/单肾切除术或马兜铃酸。我们预计缺乏T细胞受体II的小鼠损伤后G1期阻滞受损，上皮细胞存活率降低，给予palbociclib（FDA批准的G0/G1期阻滞诱导剂）可改善这一情况。体外有和没有TGF-RII的近端小管细胞将用于了解TGF-β诱导细胞周期停滞的信号通路。第二个目的是探索TGF β 1抑制上皮分化的机制以及这些变化如何影响生存。上皮分化在慢性肾损伤中的作用将使用具有Snail上皮特异性缺失的小鼠来定义，Snail是TGF-β 1下游的关键转录因子。将在体外用siRNA和构建体转染具有和不具有TGFRII的近端小管细胞，以测试Snail介导TGF-β 1依赖性去分化的假设。第三个目的是确定在肾损伤中重要的Wnt/β-连环蛋白途径如何与TGF-β相互作用以影响上皮存活。将使用遗传模型和细胞培养技术来检验TGF-β增加经典Wnt/β-catenin信号传导以通过增加去分化和G0/G1细胞周期停滞来改善上皮存活的假设。该提案采用创新的遗传学方法和一种新的战略，以定量和特定地点的方式分析伤害。这些研究将提供有价值的见解的作用 细胞周期和上皮可塑性在上皮对CKD的反应中的作用，重点是近端 肾小管是急性肾损伤的靶点，也是CKD的介质。然而，关于细胞周期、上皮可塑性和对慢性损伤的反应的信息具有超出肾脏的影响，因为大多数器官功能障碍是由慢性上皮损伤引起的。

 DESCRIPTION (provided by applicant): Chronic kidney disease (CKD) is a growing public health problem that affects 10% of Americans, increases morbidity and mortality, and imposes a huge economic burden. Persistent renal tubular injury is an important component of tubulointerstitial fibrosis (TIF), the common feature of progressive CKD of any etiology. Growth factors such as transforming growth factor- (TGF) are important determinants of how tubula epithelia respond to injury. Although excessive TGF- signaling promotes TIF, blocking TGF-using genetic tools results in increased tubular damage after mouse models of CKD, suggesting that TGF-signaling has protective effects as well. TGF-mediates many differet cellular effects including G1 arrest of cell cycle progression and epithelial de-differentiation. Tis proposal tests the hypothesis that TGFsignaling in the proximal tubule promotes survival ater chronic renal injury by inducing cell cycle arrest and epithelial de- differentiation. The first ai investigates how TGF- alters cell cycle progression and how these changes affect epithelial survival using murine models of injury and pharmacologic inducers of G1 arrest. To test this, we will use genetically modified mice that lack the TGF receptor (TRII) specifically in the proximal tubule and induce chronic kidney injury by either angiotensin/ uninephrectomy or aristolochic acid. We anticipate that mice lacking TRII have impaired G1 arrest and reduced epithelial survival after injury and that this improves with administration of palbociclib, an FDA-approved inducer of G0/G1 cell cycle arrest. Proximal tubule cells with and without TRII in vitro will be used to understand the signaling pathways through which TGF- induces cell cycle arrest. The second aim explores mechanisms whereby TGFlters epithelial differentiation nd how these changes impact survival. The role of epithelial differentiation in chronic kidney injury will be defined using mice with epithelial-specific deletion of Snail, a key transcription factor downstream of TGF- Proximal tubule cells with and without TRII will be transfected with siRNA and constructs in vitro to test the hypothesis that Snail mediates TGF--dependent de-differentiation. The third aim defines how the Wnt/-catenin pathway, important in renal injury, interacts with TGF- to affect epithelial survival. Genetic models and cell culture techniques wil be used to test the hypothesis that TGF- increases canonical Wnt/catenin signaling to improve epithelial survival through increased de-differentiation and G0/G1 cell cycle arrest. This proposal uses innovative genetic approaches and a new strategy for analyzing injury in a quantitative and location-specific way. These studies will provide valuable insights into the roles of cell cycle and epithelial plasticity in the epithelial response to CKD, focusing on the proximal tubule which is the target of acute kidney injury and a mediator of CKD. However, information about cell cycle, epithelial plasticity and response to chronic injury has implications beyond the kidney as most organ dysfunction results from chronic epithelial injury.