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）容易引发慢性肾脏病（CKD），而慢性肾脏病通常会进展为器官衰竭。 CKD 的标志之一是以细胞外基质积聚为特征的肾小管间质纤维化， 肾小管萎缩和炎症细胞浸润 AKI 的主要靶细胞是近曲小管上皮细胞。 细胞（PTEC）通过产生促炎细胞因子和细胞外基质而导致 CKD 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。 赠款旨在研究 PTEC 控制促炎症和促纤维化的分子机制 信号传导，以便设计出更有效的疗法来阻止和预防 AKI 及其进展为 CKD。 许多途径与肾纤维化的起始和进展有关，我们将重点关注 Recepteur d'Origine Nantai (RON) 和转化生长因子 β (TGF-β) 受体 II (TβRII)。 基于 TβRII 含有 5 个可磷酸化酪氨酸的证据，其中一些酪氨酸控制 为了激活 TβRII 介导的促纤维化信号传导，我们开始研究酪氨酸的作用机制 我们确定 RON 是一种能够控制 TβRII 磷酸化的主要受体酪氨酸激酶。 我们发现 RON 和 TβRII 在 PTEC 上表达，并且磷酸化 TβRII 的胞质结构域。 RON 激活导致 TβRII 酪氨酸磷酸化并增强 TβRII 介导的促炎作用（例如 此外，我们还展示了 PTEC 的治疗。 与 RON 配体 MST1 结合导致促纤维化 STAT3 的酪氨酸磷酸化和 TGF-β 的分泌， 重要的是，小鼠的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。 RON 抑制剂 Crizotinib 治疗显示 AKI 诱导的近端小管损伤减少，其特征为 酪氨酸磷酸化 RON 和 TβRII、巨噬细胞浸润、SMAD3 和 STAT3 水平降低 基于这些结果和增加酪氨酸的发现。 在患有严重 AKI 并进展的小鼠的肾脏中检测到 RON 和 TβRII 的磷酸化 对于 CKD，我们认为 PTEC 中的 RON 通过促进 1) 酪氨酸而导致肾损伤和纤维化 TβRII 磷酸化，从而增强 TβRII 介导的促炎细胞因子 (IL-18) 的产生， 促纤维化分子 (SMAD3) 的激活；以及 2) TGF-β 以 STAT3 方式分泌 诱导肌成纤维细胞分化。这笔赠款的目的旨在确定的贡献。 RON/STAT3 和 RON/TβRII 轴在肾脏疾病中的作用，并确定它们的抑制是否有益于 减缓/阻止 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 AKI 和 AKI-to-CKD 进展中的作用 我们将使用遗传和药理学方法研究表达激酶的小鼠的反应。 死亡的 RON (RONTK-/-) 和 RONTK-/- 小鼠与近端肾小管中缺乏 TβRII 的小鼠杂交，导致严重 AKI 我们将确定 IL-18 是否通过阻断来介导 RON/TβRII 促炎作用。 最后，我们将这些发现的相关性转化为 IL-18 结合蛋白的相关性。 通过研究小分子治疗小鼠对肾损伤的反应，获得更具临床相关性的设置 在目标 2 中，我们将确定 RON 导致炎症和炎症的机制。 我们将使用蛋白质测定和体外细胞培养来确定 STAT3 在 RON 介导中的作用。 我们还将鉴定 RON 和 PTEC 直接磷酸化的 TβRII 上的关键酪氨酸。 培养以确定它们在调节促炎症和促纤维化信号传导中的作用。 了解 RON/STAT3 和 RON/TβRII 轴如何调节肾损伤并探索 RON 抑制减少 TGF-β 介导的促炎症和促纤维化反应的后果将 为治疗和理想情况下预防 CKD 以及最终的终末期肾病提供了一种新方法。