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的最常见原因，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。随着亚微米囊泡从质膜脱落，微粒（MP）在健康人和啮齿动物的尿液中被发现，它们的含量和数量在细胞活化和凋亡后发生变化。这些研究的总体目标是揭示导致MP肾分泌的机制及其在糖尿病肾小管损伤中的作用。肾损伤分子-1（KIM-1）是肾近端小管损伤的尿生物标志物，最近被鉴定为参与上皮吞噬作用的清道夫受体。我们发现，肾小管KIM-1的表达显着增加，与蛋白尿和肾损伤，在Zucker糖尿病肥胖大鼠。使用大鼠和小鼠肾小管细胞原代培养物的体外研究显示，白蛋白增加了KIM-1的表达和囊泡蓄积以及其通过MP脱落的分泌。此外，KIM-1表达的增加与caspase-3活性的升高有关。有趣的是，KIM-1的敲低赋予对上皮细胞凋亡的抗性，如通过bcl-2的上调和caspase-3活化的抑制所反映的。我们进行了额外的初步研究，表明肾小管上皮细胞能够摄取MP，并且含有KIM-1（KIM-MP）的MP的内化破坏了肾小管上皮细胞单层中的正常紧密连接。因此，这些初步结果提示我们的中心假设，即“KIM-1/KIM-MP通过促进细胞损伤和凋亡而导致肾小管功能障碍”。具体而言，我们将致力于实现以下目标：目标一。为了检验肾小管表达和分泌KIM-1增加以响应白蛋白过载的假设。将进行系统分析以描述在体内动物模型和体外细胞培养模型中响应白蛋白过载和其他凋亡刺激的肾小管上皮细胞中KIM-1的表达、定位和分泌。Aim II.为了验证KIM-1/KIM-MP参与肾小管细胞损伤和凋亡的假设。KIM-1/KIM-MP对响应于白蛋白和其它凋亡刺激的肾小管细胞损伤和凋亡的作用将通过以下来确定：1）检查KIM-1的功能丧失和获得对肾小管细胞损伤和凋亡的作用，2）描绘凋亡刺激时KIM-1有助于肾小管细胞凋亡的凋亡途径，3）通过用KIM-1阳性或KIM-1阴性微粒处理正常肾小管上皮细胞单层来确定KIM-MP对靶细胞的作用。总之，目标I和II中概述的一系列研究将使我们能够对KIM-1/KIM-MP在与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。我们的研究还将揭示KIM-1/KIM-MP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.