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（阿基）易患慢性肾病（CKD），后者通常进展为器官衰竭。 CKD的标志之一是以细胞外基质积累为特征的肾小管间质纤维化， 肾小管萎缩和炎性细胞浸润。阿基的主要靶细胞是近曲小管上皮细胞 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。这个目标 格兰特的研究目的是研究PTECs控制促炎症和促纤维化的分子机制 因此，我们需要研究AKI信号传导，以便设计更有效的治疗方法来阻止和预防阿基及其进展为CKD。虽然 许多途径与肾纤维化的发生和发展有关，我们将重点关注 受体d 'Origine Nantai（罗恩）和转化生长因子β（TGF-β）受体II（TβRII）。 基于TβRII含有5个可磷酸化的酪氨酸，其中一些酪氨酸控制T β RII的磷酸化， 激活Tβ RII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。我们鉴定了罗恩作为一种主要的受体酪氨酸激酶， 磷酸化TβRII的胞质结构域。我们发现罗恩和TβRII在PTEC上表达， 罗恩激活导致TβRII酪氨酸磷酸化，并增强Tβ RII介导的促炎性（例如， IL-18分泌）和促纤维化（例如SMAD 3活化）信号传导。此外，我们还发现， 与罗恩配体MST 1的结合导致促纤维化STAT 3的酪氨酸磷酸化和TGF-β的分泌， 肌成纤维细胞分化和肾小管间质纤维化发展关键决定因素。重要的是，老鼠 用罗恩抑制剂克唑替尼治疗的患者显示AKI诱导的近端小管损伤减少，其特征在于 酪氨酸磷酸化罗恩和TβRII、巨噬细胞浸润、SMAD 3和STAT 3水平降低 活化和胶原蛋白产生。基于这些结果和发现， 在患有严重阿基的小鼠肾脏中检测到罗恩和TβRII的磷酸化， 对于CKD，我们认为PTECs中的罗恩通过促进1）酪氨酸 TβRII的磷酸化，从而增强Tβ RII介导的促炎细胞因子（IL-18）的产生， 促纤维化分子（SMAD 3）的活化;和2）TGF-β以STAT 3依赖性方式分泌， 诱导肌成纤维细胞分化。这项赠款的目的是确定以下方面的贡献： 罗恩/STAT 3和罗恩/TβRII轴在肾脏疾病中的作用，并确定其抑制是否有益于 减缓/阻止阿基及其向CKD的进展。 在目标1中，我们将研究RON介导的TβRII磷酸化在阿基和AKI向CKD进展中的作用。 使用遗传学和药理学方法。我们将研究小鼠表达一种激酶 死亡的罗恩（RONTK-/-）和RONTK-/-小鼠与近端小管中缺乏TβRII的小鼠杂交， 进展为CKD。我们将通过阻断IL-18介导的罗恩/TβRII的促炎作用， IL-18结合蛋白介导的IL-18功能。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损伤的反应， 罗恩抑制剂。在目标2中，我们将确定罗恩导致炎症的机制， 纤维化我们将使用蛋白质测定和体外细胞培养来确定STAT 3在RON介导的细胞凋亡中的作用。 TGF-β产生。我们还将鉴定TβRII上直接被罗恩和PTEC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传导中的作用。 了解罗恩/STAT 3和罗恩/TβRII轴如何调节肾损伤，并探索其作用机制。 罗恩抑制在减少TGF-β介导的促炎和促纤维化反应中的结果将 提供了一种治疗和理想地预防CKD和最终终末期肾病的新方法。