Proximal tubule Kim-1 expression modulates acute and chronic kidney injury

描述(申请人提供)：肾近端小管上皮细胞(PTCs)对肾脏的功能至关重要，也是受损肾脏病理的组成部分。它们是肾脏中对缺血和肾毒性侮辱最敏感的细胞类型。PTCs还参与调控炎症的信号通路，如分泌细胞因子、向MHC II递呈抗原以及损伤后的分解。我们已经确定Kim-1是在受损的PTCs中表达最高的蛋白。虽然Kim-1已被证明是检测肾脏损伤的一种有前途的诊断工具，但该蛋白的病理生物学功能 它在肾脏损伤中的作用尚不清楚。我们的初步数据表明，Kim-1介导的吞噬作用诱导了急性肾损伤后PTCs的自噬和细胞保护。然而，Kim-1的长期表达会导致炎症和小管损伤，类似于慢性肾脏疾病。此外，我们还发现，Kim-1介导的吞噬和自噬诱导导致抗原递呈，并且根据Kim-1表达的配体和持续时间，结果可能是促炎或抗炎。因此，我认为Kim-1介导的AKI中凋亡细胞的吞噬诱导了自噬，自噬通过MHC递呈而导致免疫调节。在AKI中，自噬的后果是细胞保护的，而在慢性状态下，PT管腔含有其他Kim-1配体，如氧化的脂类，以及较少的凋亡细胞，Kim-1介导的这些配体的摄取诱导了促炎和促纤维化反应。这是一项多学科的研究，将建立在我在细胞生物学方面的知识和研究技能的基础上，并将我的培训扩展到免疫学和老鼠遗传学等新领域。为了解决这一假设，我们将首先检验Kim-1在AKI中的保护作用是否源于其自噬诱导。我们将测试Kim-1诱导的 自噬对各种侮辱具有保护作用。然后我们将检查表达Kim-1粒细胞缺乏症小鼠的小鼠是否有改变的自噬反应。为了了解Kim-1是如何调控自噬的，Kim-1的磷酸化作用及其与GABARAP(Lc3家族成员)的相互作用在自噬诱导中的作用。其次，我们将确定Kim-1介导的内吞作用是否会导致慢性损伤。与吞噬凋亡细胞不同，氧化低密度脂蛋白等配体的内吞作用会导致线粒体碎裂和caspase激活。我们将测试转基因过表达wt Kim-1或Kim-1突变体是否会导致比过表达既不能摄取凋亡细胞也不摄取ox-LDL的突变体更大的伤害。然后，我们将测试在Kim-1介导的氧化低密度脂蛋白摄取后观察到的线粒体碎裂是否由于线粒体分裂途径的激活，以及抑制线粒体碎裂是否可以防止由Kim-1介导的氧化低密度脂蛋白内吞引起的细胞损伤。最后，我们将研究Kim-1诱导的自噬是否通过MHC递呈来调节PTC的免疫反应。我们将通过有条件地敲除MHC II来研究MHC II在AKI和CKD中的作用。然后我们将确定表达KIM-1吞噬功能缺陷突变的小鼠是否有免疫反应和自身抗体产生的改变。由于近端小管细胞也表达共刺激和共抑制因子，我们将测试Kim-1诱导的自噬是否调节这些因子的表达。这项研究的发现将阐明Kim-1在急性和慢性肾损伤中的作用，并强调调节Kim-1功能的潜在治疗益处 在肾脏损伤方面。

DESCRIPTION (provided by applicant): Renal proximal tubule epithelial cells (PTCs) are vital to the function of the kidney and are also integral to the pathology of the injured kidney. They are the most sensitive cell type in the kidney to ischemia and nephrotoxic insults. PTCs are also involved in the signaling pathways governing inflammation, such as secretion of cytokines and presentation of antigens to MHC II, and resolution following injury. We have identified Kim-1 as the most highly upregulated protein in injured PTCs. While Kim-1 has been shown to be a promising diagnostic tool for detecting kidney injury, the pathobiological function of this protein and its role in kidney injury are not known. Our preliminary data suggest that Kim-1-mediated phagocytosis induces autophagy and cytoprotection in PTCs following acute kidney injury. Prolonged expression of Kim-1, however, leads to inflammation and tubule damage, mimicking chronic kidney disease. Furthermore, we have found that Kim-1-mediated phagocytosis and autophagy induction leads to antigen presentation, and depending on the ligands and duration of Kim-1 expression, the outcome could be pro or anti-inflammatory. Therefore, I propose that Kim-1 mediated phagocytosis of apoptotic cells in AKI induces autophagy, which leads to immune modulation through MHC presentation. In AKI, the consequences of autophagy are cytoprotective while in chronic states where the PT lumen contains other Kim-1 ligands, such as oxidized lipids, and fewer apoptotic cells, Kim-1 mediated uptake of these ligands induces a pro-inflammatory, profibrotic response. This is a multidisciplinary study that will build on my knowledge and research skills in cell biology and extend my training into novel areas such as immunology and mouse genetics. To address this hypothesis we will first examine if the protective effect of Kim-1 in AKI is due to its autophagy induction. We will test if Kim- 1-induced autophagy is protective against various insults. Then we will examine if mice expressing a Kim-1 phaogocytosis deficient mouse has an altered autophagic response. To understand how Kim-1 regulates autophagy, the role of Kim-1 phosphorylation and its interaction with GABARAP (an LC3 family member) in autophagy induction. Second, we will determine if Kim-1-mediated endocytosis contributes to chronic injury. Unlike phagocytosis of apoptotic cells, endocytosis of ligands such as ox-LDL, leads to mitochondrial fragmentation and caspase activation. We will test if transgenic overexpression of wt Kim-1 or Kim-1 mutant which can take up ox-LDL but not apoptotic cells leads to greater injury than overexpression of a mutant which takes up neither apoptotic cells or ox-LDL. We will then test if the mitochondrial fragmentation observed following Kim-1-mediated uptake of ox-LDL is due to activation of the mitochondrial fission pathway and whether inhibiting mitochondrial fragmentation prevents cellular injury induced by Kim-1-mediated endocytosis of ox-LDL. Finally, we will examine if Kim-1-induced autophagy modulates the PTC immune response through MHC presentation. We will examine the role of MHC II presentation in AKI and CKD through conditional knockout of MHC II. We will then determine if mice expressing Kim-1 phagocytosis deficient mutant have an altered immune response and autoantibody production. As proximal tubule cells also express co-stimulatory and co-inhibitory factors, we will test if Kim-1 induced autophagy modulates the expression of these factors. The findings from this study will shed light on the role of Kim-1 in acute and chronic kidney injury, as well as highlight the potential therapeutic benefits of modulating Kim-1 function in kidney injury.