KIM-1, microparticles and diabetic tubular injury

KIM-1、微粒和糖尿病肾小管损伤

• 批准号: 8432030
• 负责人: Xueying Zhao
• 金额: $ 13.65万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8432030
• 关键词: Accounting; Address; Albumins; Albuminuria; Animal Model; Apoptosis; Apoptotic; Atrophic; BCL2 gene; Biological Markers; Cell Culture Techniques; Cell Survival; Cell membrane; Cells; Coculture Techniques; Data; Diabetes Mellitus; Diabetic Nephropathy; Disease Management; Disease Progression; Elements; End stage renal failure; Epithelial; Epithelial Cells; Figs - dietary; Functional disorder; Glucose; Goals; Human; In Vitro; Injury; Ischemia; Kidney; Kidney Diseases; Light; Mediator of activation protein; Modeling; Molecular; Mus; Pathogenesis; Pathway interactions; Pattern; Phagocytosis; Pharmaceutical Preparations; Pilot Projects; Proteins; Proteinuria; Rattus; Research; Resistance; Rodent; Role; Series; Signal Transduction; Stimulus; Stress; Testing; Tight Junctions; Tubular formation; Up-Regulation; Urine; Vesicle; base; caspase-3; cell injury; diabetic; diabetic rat; gain of function; in vivo; insight; monolayer; novel therapeutics; oxidized low density lipoprotein; prevent; rat KIM-1 protein; response; scavenger receptor; submicron; uptake; urinary

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.

描述（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，肾小管萎缩是疾病进展的敏感预测因子。随着亚微米囊泡从质膜脱落，健康人和啮齿动物的尿液中发现了微粒（MP），它们的含量和数量随着细胞激活和凋亡而改变。拟议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) 是肾近端肾小管损伤的尿液生物标志物，最近被确定为参与上皮吞噬作用的清道夫受体。我们发现，在 Zucker 糖尿病肥胖大鼠中，KIM-1 的肾小管表达显着增加，与蛋白尿和肾损伤相关。使用大鼠和小鼠肾小管细胞原代培养物进行的体外研究表明，白蛋白可增加 KIM-1 的表达和囊泡积累，并通过 MP 脱落增加其分泌。此外，KIM-1 表达的增加与 caspase-3 活性的升高相关。有趣的是，KIM-1 的敲低赋予了对上皮细胞凋亡的抵抗力，这反映在 bcl-2 的上调和 caspase-3 激活的抑制上。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单层中的正常紧密连接。因此，这些初步结果促使我们提出中心假设：“KIM-1/KIM-MPs 通过促进细胞损伤和凋亡而导致肾小管功能障碍”。具体来说，我们将实现以下目标： 目标 I. 检验 KIM-1 的肾小管表达和分泌因白蛋白过载而增加的假设。将进行系统分析，以描述体内动物模型和体外细胞培养模型中肾小管上皮细胞响应白蛋白超载和其他凋亡刺激而表达、定位和分泌的 KIM-1。目标二。检验 KIM-1/KIM-MP 导致肾小管细胞损伤和凋亡的假设。 KIM-1/KIM-MP 对白蛋白和其他凋亡刺激反应中的肾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 功能丧失和功能获得对肾小管细胞损伤和凋亡的影响，2) 描述 KIM-1 在凋亡刺激后促进肾小管细胞凋亡的凋亡途径，3) 确定 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，将 KIM-MPs 作用于靶细胞。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。我们的研究还将阐明 KIM-1/KIM-MP 作为肾脏疾病管理的有吸引力的靶标。