Sphingolipids in Acute Kidney Injury and Disease Progression

当前提案的总体目标是了解靶向鞘脂的机制， 肾脏纤维化途径可以预防或减轻进行性肾纤维化。美国约有20- 40百万人拥有肾脏 我们迫切需要新的治疗方法。靶向鞘脂途径是重要的 生物学研究领域，将加强新的治疗干预措施，用于治疗进行性 肾纤维化鞘氨醇1-磷酸（S1 P）是S1 P受体（S1 PR）的天然配体。 并且存在于细胞外和细胞内区室中。细胞外S1 P的作用是通过 通过S1 PR 1 -5，其由多种细胞类型差异表达。细胞内S1 P由 操纵S1 P合成、降解或输出。细胞内S1 P是一种关键的信号分子， 多功能的角色，取决于它的划分。细胞内S1 P通过磷酸化产生 两种鞘氨醇激酶（Sphk 1和Sphk 2）。Sphk 1定位于细胞质，Sphk 2 定位于细胞核、线粒体和内质网。我们观察到肾组织 与Sphk 1-/-或WT小鼠相比，Sphk 2-/-小鼠中的纤维化，以及 周细胞/血管周细胞。Sphk 2产生的S1 P结合并抑制组蛋白脱乙酰酶（HDAC）活性 并增强基因表达。最近，Spns 2已被鉴定为S1 P输出途径; 在培养的肾细胞中，S1 P转运增加细胞质S1 P并抑制纤维化因子的产生， 和Spns 2缺陷型小鼠与炎性疾病状况有关。Spns 2在大肠杆菌中高表达， 近端小管、内皮细胞和周细胞。最后，我们发现，一个领先的Spns 2抑制剂衰减阿基。这些 研究结果使我们假设，S1 P的房室控制是进行性 肾脏纤维化。目的1将检验核Sphk 2缺陷保护小鼠免于肾纤维化的假设。 我们将确定Sphk 2的酶活性或核定位是否是纤维化所必需的。 目的2将检验Spns 2抑制或Spns 2缺乏保护小鼠免于进行性肾损害的假设。 纤维化我们将确定1）整体Spns 2缺陷小鼠是否受到保护免于纤维化，以及2）经治疗的小鼠是否受到保护。 与Spns 2抑制剂联合使用，可以防止纤维化。目标3.将检验控制肾脏的假设 S1 P的实质输出在减弱纤维化中是关键的。我们假设这种保护作用是由于 近端小管细胞、内皮细胞或周细胞中S1 P输出减少。我们会产生周细胞， 内皮和近端小管Spns 2-/-小鼠，并确定对纤维化的影响。我们将进行体外研究 并确定S1 P周细胞、内皮细胞或近曲小管细胞的隔室控制的作用 控制周细胞向肌成纤维细胞转变的因子。我们相信，这些研究将进一步推动 了解进行性肾纤维化的发病机制，并为发展 新的治疗剂。

The overall goal of the current proposal is to understand the mechanisms by which targeting the sphingolipid pathway can prevent or attenuate progressive kidney fibrosis. ~20-40M people in the United States have kidney diseases and new treatments are urgently needed. Targeting the sphingolipid pathway serves as an important area for biological research that will undergird novel therapeutic interventions for the treatment of progressive kidney fibrosis. Sphingosine 1-phosphate (S1P) is the naturally occurring ligand of the S1P receptors (S1PR) and is present in both extracellular and intracellular compartments. The effects of extracellular S1P are mediated through S1PR1-5, which are differentially expressed by a variety of cell types. Intracellular S1P is controlled by manipulating S1P synthesis, degradation, or export. Intracellular S1P is a key signaling molecule that has multifunctional roles, depending on its compartmentalization. Intracellular S1P is generated by phosphorylation of sphingosine by two sphingosine kinases (Sphk1 and Sphk2). Sphk1 is localized to the cytoplasm and Sphk2 localized to the nucleus, mitochondria, and endoplasmic reticulum. We observed markedly attenuated renal fibrosis in Sphk2-/- mice compared to Sphk1-/- or WT mice and in mice with tissue specific deletion of Sphk2 in pericyte/perivascular cells. S1P generated by Sphk2 binds to and inhibits histone deacetylase (HDAC) activity and enhances gene expression. Recently Spns2 has been identified as an S1P export pathway; inhibition of S1P transport increases cytoplasmic S1P and inhibits production of fibrogenic factors in cultured kidney cells, and Spns2 deficient mice are associated with inflammatory disease conditions. Spns2 is highly expressed in proximal tubule, endothelium and pericytes. Lastly we found that a lead Spns2 inhibitor attenuated AKI. These findings lead us to hypothesize that compartmental control of S1P is a critical determinant of progressive kidney fibrosis. Aim 1 will test the hypothesis that nuclear Sphk2-deficiency protects mice from kidney fibrosis. We will determine whether the enzymatic activity or the nuclear localization of Sphk2 are necessary for fibrosis. Aim 2 will test the hypothesis that Spns2 inhibition or Spns2 deficiency protects mice from progressive kidney fibrosis. We will determine whether 1) global Spns2 deficient mice are protected from fibrosis and 2) mice treated with a lead Spns2 inhibitor, are protected from fibrosis. Aim 3. Will test the hypothesis that control of kidney parenchymal export of S1P is critical in attenuating fibrosis. We hypothesize that the protective effect is due to decreased export of S1P in proximal tubule cells, endothelial cells or pericytes. We will generate pericyte, endothelial and proximal tubule Spns2-/- mice and determine the effect on fibrosis. We will perform in vitro studies and determine the role of compartmental control of S1P pericytes, endothelial cells or proximal tubule cells factors that control pericyte to myofibroblast transition. We believe that these studies will lead to further understanding of the pathogenesis of progressive kidney fibrosis and provide the foundation for the development of novel therapeutics agents.