Sphingolipids in Acute Kidney Injury and Disease Progression

 描述(由申请人提供)：进行性肾脏疾病和终末期肾病与高发病率和死亡率有关。不管损伤的原因是什么，都会有导致间质纤维化的典型反应。一个关键特征是激活产生细胞外基质的肌成纤维细胞。目前的治疗主要集中在非特异性或支持性治疗上。了解纤维化的机制将导致新的治疗方法。在目前的供资周期中，我们加深了对鞘脂在AKI中的作用的理解。鞘氨醇1-磷酸(S1P)是一种多功能溶血磷脂，参与细胞生长和存活、淋巴细胞运输和血管稳定等多种功能。 对免疫系统和肾脏损伤有深远的影响。S1P是鞘氨醇被两种不同亚细胞定位的鞘氨醇激酶亚型(SphK1和SphK2)磷酸化的产物。SphK1是细胞质的，而SphK2定位于细胞核、线粒体和内质网，表明1-磷酸鞘氨醇具有细胞内/核内信号转导作用。我们观察到，与Sphk1/-或WT小鼠相比，Sphk2-/-小鼠的肾脏纤维化显著减轻，组织中干扰素-显著升高。这些发现促使我们将重点放在SphK2的特定作用上，并确定核内SphK2是否调节组织纤维化。我们的初步数据为目前的建议提供了一个框架：1)在两种纤维化模型(叶酸诱导的肾病(FA-N)和单侧缺血再灌注后的疾病进展)中，我们发现缺乏SphK2的小鼠显著减少了纤维化和高组织表达的干扰素。2)延迟给药一种新的SphK2抑制剂可以保护小鼠免受纤维化，3)过继转移SphK2缺陷的T细胞，它产生高水平的干扰素，保护WT小鼠免受纤维化的影响。4)对Sphk2-/-培养的成纤维细胞的刺激抵抗肌成纤维细胞的转化；5)结合基因芯片数据、H3K27ac芯片序列和高通量测序，我们已经确定了可能抑制Sphk2-/-小鼠肾脏纤维化的组蛋白乙酰化的候选基因。我们提出了一系列的体外和体内研究，以更好地了解SphK2在肾脏纤维化中的作用。我们的总体假设是：1)从AKI到纤维化的进展(或AKI后的修复和恢复过程)受SphK2调控；2)SphK2的缺失或抑制有助于抵抗纤维化。目的1.验证SphK2药物抑制或SphK2基因缺陷减轻或逆转肾纤维化的假说。目的2.通过对局部间质干扰素的调节，验证药物抑制SphK2阻断纤维化或SphK2基因缺陷小鼠抵抗肾纤维化的假说。目的3.验证SphK2介导表遗传调控导致肾纤维化关键基因表达的假说。这些研究将帮助我们了解S1P如何调节纤维化/修复，但它们也有可能发现对疾病进展的各个方面至关重要的基因，这些基因可能成为治疗的靶点。

 DESCRIPTION (provided by applicant): Progressive kidney disease and ESRD are associated with high morbidity and mortality. Regardless of the cause of injury there is a stereotypical response leading to interstitial fibrosis. A key feature is the activation of extracellular matrix-producing myofibroblasts. Current therapies focus on nonspecific or supportive therapy. Understanding mechanisms of fibrosis will lead to new therapies. During the course of the current funding cycle we advanced our understanding of the role of sphingolipids in AKI. Sphingosine 1-phosphate (S1P), a pleiotrophic lysophospholipid that is involved in diverse functions such as cell growth and survival, lymphocyte trafficking, and vascular stability, has profound effects on the immune system and kidney injury. S1P is the product of sphingosine phosphorylation by two sphingosine kinase isoforms (SphK1 and SphK2) that have different subcellular localizations. Whereas SphK1 is cytoplasmic, SphK2 is localized in the nucleus, mitochondria and endoplasmic reticulum pointing to an intracellular/intranuclear signaling role of sphingosine 1-phosphate. We observed that Sphk2-/- mice had markedly attenuated renal fibrosis compared to Sphk1-/- or WT mice and marked tissue elevation of interferon gamma (IFN). These findings led us to focus our renewal on the specific role of SphK2 and determine whether intranuclear SphK2 regulates tissue fibrosis. Our preliminary data provide a framework for the current proposal: 1) In 2 models of fibrosis (folic acid-induced nephropathy (FA-N) and disease progression after unilateral IRI) we found that mice deficient in SphK2 have markedly reduced fibrosis and high tissue expression of IFN2) delayed administration of a novel inhibitor of SphK2 protects mice from fibrosis, 3) adoptive transfer of SphK2-deficient T cells, which produce high levels of IFN, protects WT mice from fibrosis, 4) stimulation of fibroblasts cultured from Sphk2-/- are resistant to myofibroblast transformation and 5) combining microarray data and ChIP-seq of H3K27ac and high-throughput sequencing we have identified candidate genes with histone acetylations that may suppress fibrosis in kidneys of Sphk2-/- mice. We propose a series of in vitro and in vivo studies to better understand the role of SphK2 in kidney fibrosis. Our overall hypothesis is that: 1) the progression from AKI to fibrosis (or the process of repair and recovery after AKI) is regulated by SphK2 and 2) the absence or inhibition of SphK2 contributes to resistance to fibrosis. Aim 1. To test the hypothesis that pharmacological inhibition of SphK2 or genetic deficiency of SphK2 attenuates or reverses kidney fibrosis. Aim 2. To test the hypothesis that pharmacological inhibition of SphK2 blocks fibrosis or mice deficient of SphK2 are resistant to kidney fibrosis through regulation of local interstitial IFN. Aim 3. To test the hypothesis that SphK2 mediates epigenetic regulation of expression of key genes that can cause kidney fibrosis. These studies will help us understand how S1P regulates fibrosis/repair but they also have the potential to uncover genes important for various aspects of disease progression that may serve as targets for therapy.