KIM-1, microparticles and diabetic tubular injury

描述（由申请人提供）：糖尿病肾病目前是终末期肾病最常见的病因，占40-50% 1，细胞凋亡已被证明是肾小管萎缩的重要机制，是疾病进展的敏感预测因子。当亚微米囊泡从质膜脱落时，在健康人类和啮齿动物的尿液中发现微颗粒（MPs），它们的含量和数量在细胞激活和凋亡后发生改变。提出的研究的总体目标是揭示导致MPs肾脏分泌的机制及其在糖尿病小管损伤中的作用。肾损伤分子-1 （KIM-1）是肾近端小管损伤的尿液生物标志物，最近被发现是一种参与上皮吞噬的清除受体。我们发现，在Zucker糖尿病脂肪大鼠中，肾小管中KIM-1的表达显著增加，与蛋白尿和肾损伤有关。利用大鼠和小鼠小管细胞原代培养的体外研究表明，白蛋白增加了KIM-1的表达和囊泡积聚，并通过MP脱落分泌。此外，这种KIM-1表达的增加与caspase-3活性的升高有关。有趣的是，KIM-1的下调可以通过上调bcl-2和抑制caspase-3激活来抵抗上皮细胞凋亡。我们进行了进一步的初步研究，表明小管上皮细胞能够吸收MPs，并且含有KIM-1的MPs的内化破坏了小管上皮单层的正常紧密连接。因此，这些初步结果支持了我们的中心假设，即“KIM-1/KIM- MPs通过促进细胞损伤和凋亡来促进小管功能障碍”。具体来说，我们将解决以下目的：目的一：验证小管中KIM-1的表达和分泌在白蛋白超载的情况下增加的假设。在体内动物模型和体外细胞培养模型中，我们将系统分析KIM-1在小管上皮细胞中对白蛋白超载和其他凋亡刺激的表达、定位和分泌。目的二世。验证KIM-1/KIM-MPs参与小管细胞损伤和凋亡的假说。在白蛋白和其他凋亡刺激下，KIM-1/KIM-MPs对小管细胞损伤和凋亡的影响将通过以下方式确定：1)检查KIM-1的功能丧失和功能获得对小管细胞损伤和凋亡的影响；2)描述KIM-1在凋亡刺激下促进小管细胞凋亡的凋亡途径。3)通过用KIM-1阳性或KIM-1阴性微粒处理正常小管上皮细胞单层，确定KIM-MPs对靶细胞的影响。总之，Aims I和II中概述的一系列研究将使我们能够为KIM-1/KIM-MPs在糖尿病肾病相关的肾小管损伤和功能障碍中的功能作用提供新的见解。我们的研究还将阐明KIM-1/KIM-MPs作为肾脏疾病管理的有吸引力的靶点。

DESCRIPTION (provided by applicant): Diabetic nephropathy is now the most prevalent cause of end stage renal disease, accounting for 40-50% 1, and apoptosis has been shown to be an important mechanism for tubular atrophy, a sensitive predictor of disease progression. As the submicron vesicles shed from plasma membranes, microparticles (MPs) are found in the urine of healthy humans and rodents and their contents and amounts are altered following cell activation and apoptosis. The overall goal of the proposed studies is to uncover the mechanisms leading to renal secretion of MPs and their role in tubular injury in diabetes. Kidney injury molecule-1 (KIM-1) is a urinary biomarker for renal proximal tubular damage and has been recently identified as a scavenger receptor involved in epithelial phagocytosis. We found that tubular expression of KIM-1 was dramatically increased, in association with proteinuria and kidney injury, in Zucker diabetic fatty rats. In vitro studies using primary cultures of rat and mouse tubular cells revealed that albumin increased expression and vesicular accumulation of KIM-1 as well as its secretion via MP shedding. Moreover, this increase in KIM-1 expression was associated with an elevation of caspase-3 activity. Interestingly, knockdown of KIM-1 confers resistance to epithelial apoptosis as reflected by an up-regulation of bcl-2 and suppression of caspase-3 activation. We conducted additional pilot study indicating that tubular epithelial cells were capable of uptaking MPs and that the internalization of MPs containing KIM-1 (KIM-MPs) disrupted normal tight junction in tubular epithelial monolayer. Therefore, those preliminary results prompted our central hypothesis stating that "KIM-1/KIM- MPs contribute to tubular dysfunction by promoting cell injury and apoptosis". Specifically, we will address the following aims: Aim I. To test the hypothesis that tubular expression and secretion of KIM-1 are increased in response to albumin overload. Systematic analyses will be conducted to delineate expression, localization and secretion of KIM-1 in tubular epithelial cells in response to albumin overload and other apoptotic stimuli in both in vivo animal model and in vitro cell culture model. Aim II. To test the hypothesis that KIM-1/KIM-MPs contribute to tubular cell injury and apoptosis. The effects of KIM-1/KIM-MPs on tubular cell injury and apoptosis in response to albumin and other apoptotic stimuli will be determined by 1) examining the effects of loss- and gain-of-function of KIM-1 on tubular cell injury and apoptosis, 2) delineating the apoptotic pathways by which KIM-1 contributes to tubular cell apoptosis upon apoptotic stimulation, 3) determining the effects of KIM-MPs on the target cells by treating normal tubular epithelial cell monolayer with KIM-1 positive or KIM-1 negative microparticles. Together, the series of studies outlined in Aims I and II will enable us to provide new insights into the functional role of KIM-1/KIM-MPs in tubular injury and dysfunction associated with diabetic kidney disease. Our studies will also shed light on KIM-1/KIM-MPs as attractive targets for kidney disease management.