Molecular Mechanisms of Kidney Fibrosis

肾脏纤维化的分子机制

• 批准号: 10480325
• 负责人: AMBRA POZZI
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2026-09-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10480325
• 关键词: Acute; Acute Renal Failure with Renal Papillary Necrosis; Atrophic; Binding; Biological Assay; Cell Culture Techniques; Cells; Chronic Kidney Failure; Chronic Phase; Collagen; Collagen Receptors; Cytoplasmic Tail; Data; Deposition; Development; Disease Progression; End stage renal failure; Epithelial Cells; Epithelium; Extracellular Matrix; Fibroblasts; Fibrosis; Genetic; Goals; Grant; Homeostasis; IL18 gene; In Vitro; Infiltration; Inflammation; Inflammatory; Injury; Injury to Kidney; Integrins; Kidney; Kidney Diseases; Ligands; MADH3 gene; Macrophage; Mediating; Mediator; Modeling; Molecular; Morbidity - disease rate; Mus; Myofibroblast; Organ; Organ failure; Pathogenesis; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Play; Prevention; Process; Production; Profibrotic signal; Protein Tyrosine Kinase; Protein Tyrosine Phosphatase; Proteins; Receptor Protein-Tyrosine Kinases; Receptor Serine/Threonine Kinase; Regulation; Renal function; Role; STAT3 gene; Signal Transduction; T-cell protein tyrosine phosphatase; Tail; Testing; Transforming Growth Factor Beta 2; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; Translating; Tubular formation; Tyrosine; Tyrosine Phosphorylation; Veterans; cell type; clinically relevant; constitutive expression; crizotinib; cytokine; defined contribution; design; effective therapy; in vivo; inhibitor; injured; interleukin-18 binding protein; interstitial; kidney epithelial cell; kidney fibrosis; loss of function; mortality; novel; novel strategies; overexpression; pharmacologic; prevent; receptor; response; small molecule

Acute kidney injury (AKI) predisposes to chronic kidney disease (CKD) which often progresses to organ failure. One of the hallmarks of CKD is tubulointerstitial fibrosis characterized by extracellular matrix accumulation, tubular atrophy and inflammatory cell infiltration. The main cells targeted by AKI are proximal tubule epithelial cells (PTECs) that contribute to CKD by producing pro-inflammatory cytokines and extracellular matrix and by stimulating myofibroblast differentiation of surrounding fibroblasts, leading to interstitial fibrosis. The goal of this grant is to investigate the molecular mechanisms whereby PTECs control pro-inflammatory and pro-fibrotic signaling in order to devise more effective therapies to halt and prevent AKI and its progression to CKD. Although many pathways have been implicated in both initiation and progression to kidney fibrosis, we will focus on Recepteur d'Origine Nantai (RON) and transforming growth factor beta (TGF-β) receptor II (TβRII). Based on the evidence that TβRII contains 5 phosphorylatable tyrosines and some of these tyrosines control activation of TβRII-mediated profibrotic signaling, we started to investigate the mechanisms whereby tyrosine phosphorylation of TβRII is controlled. We identified RON as a major receptor tyrosine kinase able to phosphorylate the cytoplasmic domain of TβRII. We show that RON and TβRII are expressed on PTECs and RON activation leads to TβRII tyrosine phosphorylation and enhances TβRII-mediated pro-inflammatory (e.g. IL-18 secretion) and pro-fibrotic (e.g. SMAD3 activation) signaling. In addition, we show that treatment of PTECs with the RON ligand MST1 leads to tyrosine phosphorylation of pro-fibrotic STAT3 and secretion of TGF-β, a key determinant in myofibroblast differentiation and development of tubulointerstitial fibrosis. Importantly, mice treated with the RON inhibitor Crizotinib show reduced AKI-induced proximal tubule injury characterized by decreased levels of tyrosine phosphorylated RON and TβRII, macrophage infiltration, SMAD3 and STAT3 activation, and collagen production. Based on these results and the finding that increased tyrosine phosphorylation of RON and TβRII is detected in the kidneys of mice subjected to severe AKI that progresses to CKD, we propose that RON in PTECs contributes to kidney injury and fibrosis by promoting 1) tyrosine phosphorylation of TβRII thus enhancing TβRII-mediated production of pro-inflammatory cytokines (IL-18) and activation of pro-fibrotic molecules (SMAD3); and 2) TGF-β secretion in a STAT3 dependent manner thus inducing myofibroblast differentiation. The aims of this grant are designed to define the contribution of RON/STAT3 and RON/TβRII axes in kidney disease and to determine if their inhibition is beneficial in slowing/halting AKI and its progression to CKD. In Aim 1 we will study the role of RON-mediated phosphorylation of TβRII in AKI and AKI-to-CKD progression using a genetic and pharmacological approach. We will investigate the response of mice expressing a kinase dead RON (RONTK-/-) and RONTK-/- mice crossed to mice lacking TβRII in proximal tubules to severe AKI that progresses to CKD. We will determine whether IL-18 mediates RON/TβRII pro-inflammatory action by blocking IL18-mediated function with IL-18-binding protein. Finally, we will translate the relevance of these findings to a more clinically relevant setting by investigating the response to kidney injury in mice treated with a small molecule RON inhibitor. In Aim 2 we will determine the mechanisms whereby RON contributes to inflammation and fibrosis. We will use protein assays and in vitro cell cultures to determine the role of STAT3 in RON-mediated TGF-β production. We will also identify the key tyrosines on TβRII directly phosphorylated by RON and PTEC cultures to determine their role in regulating pro-inflammatory and pro-fibrotic signaling. Understanding how the RON/STAT3 and RON/TβRII axes regulate kidney injury and exploring the consequences of RON inhibition in reducing TGF-β-mediated pro-inflammatory and pro-fibrotic responses will offer a novel approach for the treatment and, ideally, prevention of CKD and ultimately end stage kidney disease.

急性肾损伤(AKI)易导致慢性肾脏病(CKD)，慢性肾脏病往往进展为器官衰竭。 慢性肾脏病的特征之一是以细胞外基质堆积为特征的肾小管间质纤维化， 肾小管萎缩、炎性细胞浸润。AKI靶向的主要细胞是近端小管上皮 细胞(PTECs)通过产生促炎细胞因子和细胞外基质以及通过 刺激周围成纤维细胞的肌成纤维细胞分化，导致间质纤维化。这样做的目的是 格兰特将研究PTECs控制促炎和促纤维化的分子机制 以设计更有效的治疗方法来阻止和防止AKI及其进展为CKD。虽然 许多途径都与肾纤维化的发生和发展有关，我们将重点关注 南台受体(RON)和转化生长因子-β(β)受体II(TβRII)。 基于TβRII含有5种可磷酸化酪氨酸的证据，以及其中一些酪氨酸控制 激活TβRII介导的促纤维化信号，我们开始研究酪氨酸 TβRII的磷酸化受到控制。我们发现RON是一种主要的受体酪氨酸激酶，能够 使TβRII的胞质结构域磷酸化。我们发现RON和TβRII在PTEC和 RON激活导致TβRII酪氨酸磷酸化，增强TβRII介导的促炎作用(例如 IL-18分泌)和促纤维化(如Smad3激活)信号转导。此外，我们还展示了对PTECs治疗 随着RON配体MST1导致促纤维化STAT3的酪氨酸磷酸化和转化生长因子-β的分泌，a 肌成纤维细胞分化和肾小管间质纤维化发展的关键决定因素。重要的是，老鼠 使用RON抑制剂Crizotinib可减少AKI诱导的近端小管损伤，其特征是 酪氨酸磷酸化RON和TβRII、巨噬细胞浸润、Smad3和STAT3水平降低 活化和胶原蛋白的产生。根据这些结果和发现增加的酪氨酸 在进展性急性肾损伤小鼠肾脏中检测到RON和T-βRII的磷酸化 对于慢性肾脏病，我们认为PTECs中的RON通过促进酪氨酸参与了肾脏损伤和纤维化 TβRII的磷酸化从而增强TβRII介导的促炎细胞因子(IL-18)和 促纤维化分子的激活(Smad3)；以及2)转化生长因子-β的分泌依赖于STAT3 诱导肌成纤维细胞分化。这笔赠款的目的是为了定义 RON/STAT3和RON/TβRII轴在肾脏疾病中的作用及其抑制是否有益于 减缓/停止AKI及其向CKD的进展。 在目标1中，我们将研究RON介导的TβRII磷酸化在急性心肌梗死和急性心肌梗死向慢性肾脏病进展中的作用 使用遗传和药理学方法。我们将研究表达一种激酶的小鼠的反应 死亡RON(RONTK-/-)和RONTK-/-小鼠与严重AKI近端小管缺乏TβRII的小鼠杂交 进展为慢性肾脏病。我们将确定IL-18是否通过阻断RON/TβRII介导促炎作用 IL-18介导的功能与IL-18结合蛋白。最后，我们将把这些发现的相关性转化为 通过研究小分子治疗小鼠对肾脏损伤的反应，更具临床相关性 RON抑制剂。在目标2中，我们将确定RON促进炎症和 纤维化症。我们将使用蛋白质分析和体外细胞培养来确定STAT3在RON介导的过程中的作用 转化生长因子-β的产生。我们还将确定TβRII上由RON和PTEC直接磷酸化的关键酪氨酸 培养以确定它们在调节促炎和促纤维化信号中的作用。 了解RON/STAT3和RON/TβRII轴如何调节肾损伤，并探索 抑制RON在减少转化生长因子-β介导的促炎和促纤维化反应中的后果 为治疗慢性肾脏病并最终预防终末期肾病提供了一种新的方法，最好是预防。