TGF-beta Pathways that Protect Epithelia in Chronic Renal Injury

 描述(申请人提供)：慢性肾脏疾病(CKD)是一个日益严重的公共卫生问题，影响着10%的美国人，增加了发病率和死亡率，并施加了巨大的经济负担。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是任何病因的进展性CKD的共同特征。转化生长因子-(转化生长因子-)等生长因子是肾小管上皮细胞对损伤反应的重要决定因素。尽管过度的转化生长因子-信号促进了肾间质纤维化，但使用基因工具阻断转化生长因子-会导致慢性肾脏病小鼠模型肾小管损伤的增加，提示转化生长因子-信号也具有保护作用。转化生长因子-介导了许多不同的细胞效应，包括G1期细胞周期停滞和上皮细胞去分化。TIS方案验证了近端肾小管转化生长因子信号通过诱导细胞周期停滞和上皮去分化而提高慢性肾损伤患者存活率的假说。第一个人工智能研究转化生长因子-是如何改变细胞周期进程的，以及这些变化是如何影响上皮细胞存活的，使用的是小鼠损伤模型和药物诱导的G1期停滞。为了验证这一点，我们将使用转基因小鼠，这些小鼠在近端小管上缺乏转化生长因子受体(TRII)，并通过血管紧张素/单肾切除术或马兜铃酸诱导慢性肾脏损伤。我们预计，缺乏TRII的小鼠损伤后G1期停滞和上皮细胞存活率下降，而给予FDA批准的G0/G1细胞周期停滞诱导剂Palbociclib可以改善这一点。近端肾小管上皮细胞加入和不加入TRII将被用来了解转化生长因子-诱导细胞周期停滞的信号通路。第二个目的是探讨转化生长因子诱导上皮细胞分化的机制以及这些变化如何影响存活率。上皮分化在慢性肾损伤中的作用将通过上皮特异性缺失Snail的小鼠来确定，Snail是转化生长因子-近端小管下游的关键转录因子，有和没有T-RII的近端小管细胞将被转染小干扰RNA并在体外构建，以检验Snail介导转化生长因子-依赖的去分化的假说。第三个目标定义了在肾脏损伤中起重要作用的Wnt/-catenin通路如何与转化生长因子-相互作用影响上皮细胞的存活。遗传模型和细胞培养技术将被用来检验这一假说，即转化生长因子-通过增加去分化和G0/G1细胞周期停滞来增加典型的Wnt/连环蛋白信号，从而改善上皮细胞的存活。这项建议使用了创新的遗传方法和一种新的策略，以定量和特定位置的方式分析损伤。这些研究将为了解这些角色提供有价值的见解 细胞周期和上皮可塑性在慢性肾脏病上皮反应中的作用，侧重于近端 肾小管是急性肾损伤的靶点，也是慢性肾脏病的介质。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越肾脏的意义，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.