KIDNEY INJURY MOLECULE-1 IN EPITHELIAL REPAIR

描述（由申请人提供）：肾损伤分子-1（KIM-1）在以上皮细胞去分化为特征的各种状态下的近端肾小管上皮细胞中强烈上调：缺血、毒性肾损伤、多囊肾病和肾细胞癌。它比任何其他已知的蛋白质在肾损伤中上调更多。KIM-1蛋白是一种I型细胞膜糖蛋白，含有细胞外免疫球蛋白样和粘蛋白结构域，表明在细胞-细胞和/或细胞-基质相互作用中的功能作用。我们已经克隆了人、小鼠和大鼠KIM-1，并产生了其单克隆和多克隆抗体。KIM-1在体外邻近机械“伤口”的MDCK细胞中上调。KIM-1的胞外域被切割，并在急性肾小管坏死或肾细胞癌患者的尿液中发现。LLC-PK 1上皮细胞中KIM-1的基因转移导致具有间充质形态的细胞数量增加，并赋予在软琼脂中的生长。该提案的目标是表征KIM-1在上皮细胞粘附和分化过程中的功能作用。我们假设KIM-1减少细胞与细胞的粘附，增加细胞与基质的粘附，并增强上皮细胞向间充质的转化。在具体目标1中，我们将表征KIM-1对上皮细胞的细胞-细胞和细胞-基质相互作用的影响。在初步实验中，我们发现表达KIM-1的LLC-PK 1细胞的细胞-细胞聚集减少。过表达KIM-1的CHO细胞“分散”在各种基质底物上。KIM-1表达导致E-钙粘蛋白的丢失和细胞-基质粘附的增加。ERM蛋白，整合素，Rho，整合素连接和丝裂原活化蛋白（MAP）激酶作为KIM-1对细胞-细胞和细胞-基质粘附作用的效应物的作用将被探索。将评价KIM-1的细胞外和细胞内结构域以及KIM-1的细胞内结构域的酪氨酸磷酸化在KIM-1对粘附的作用中的贡献。在第二个具体目标中，我们将分析KIM-1在与肾小管上皮细胞去分化相关的过程中的作用，这些细胞对于上皮细胞的修复具有积极和潜在的负面影响。我们将探索TGF-β在增强KIM-1对细胞分化和上皮向间充质转化的作用中的作用。提出了一种机制，涉及ERM蛋白，Rho，整合素连接和MAP家族激酶以及α-连环蛋白和转录因子Snail，在KIM-1对肾上皮细胞分化状态的影响。在多囊肾病模型中，表达KIM-1的近端小管位于肾纤维化增加的区域，我们将探讨KIM-1表达对基质蛋白产生和细胞外基质重塑的影响。了解KIM-1的功能将为了解该蛋白在肾脏损伤和修复过程中的作用提供重要的见解，并可能将KIM-1确定为不仅用于急性和慢性肾脏疾病而且用于上皮细胞恶性转化的重要治疗靶点。

DESCRIPTION (provided by applicant): Kidney injury molecule-1 (KIM-1) is strongly upregulated in proximal tubular epithelial cells in various states characterized by epithelial cell dedifferentiation: ischemia, toxic renal injury, polycystic kidney disease and renal cell carcinoma. It is upregulated more than any other known protein with renal injury. KIM-1 protein is a Type I cell membrane glycoprotein containing extracellular immunoglobulin-like and mucin domains suggesting functional roles in cell-cell and/or cell-matrix interactions. We have cloned, and generated monoclonal and polyclonal antibodies to, the human, mouse and rat KIM-1. KIM-1 is upregulated in vitro in MDCK cells adjacent to a mechanical "wound". The ectodomain of KIM-1 is cleaved and found in urine of patients with acute tubular necrosis or renal cell carcinoma. Gene transfer of KIM-1 in LLC-PK1 epithelial cells results in an increased number of cells with mesenchymal morphology and confers growth in soft agar. The goal of this proposal is to characterize the functional role of KIM-1 during the processes of adhesion and differentiation of epithelial cells. We hypothesize that KIM-1 reduces cell-cell and increases cell-matrix adhesion and potentiates epithelial to mesenchymal transition. In Specific Aim 1, we will characterize the effects of KIM-1 on cell-cell and cell-matrix interactions of epithelial cells. In pilot experiments, we found decreased cell-cell aggregation of LLC-PK1 cells expressing KIM-1. CHO cells overexpressing KIM-1 "scatter" on various matrix substrates. KIM-1 expression results in loss of E-cadherin and increased cell-matrix adhesion. The roles of ERM proteins, integrins, Rho, integrin-linked and mitogen-activated protein (MAP) kinases as effectors of KIM-1 actions on cell-cell and cell-matrix adhesion will be explored. The contributions of the extracellular and intracellular domains of KIM-1 and tyrosine phosphorylation of the intracellular domain of KIM-1 in KIM-l's effects on adhesion will be evaluated. In the second specific aim, we will analyze the role of KIM-1 in the processes associated with dedifferentiation of renal tubular epithelial cells that are important for repair of the epithelium with both positive and potentially negative consequences. We will explore the role of TGF-beta in potentiating the effects of KIM-1 on cell differentiation and epithelial to mesenchymal transition. A mechanism is proposed that implicates ERM proteins, Rho, integrin-linked and MAP family kinases as well as a-catenin and the transcription factor Snail, in the effect of KIM-1 on the differentiation status of the renal epithelial cell. KIM-1-expressing proximal tubules localize in areas of the kidney with increased fibrosis in a model of polycystic kidney disease and we will explore if there is an effect of KIM-1 expression on matrix protein production and extracellular matrix remodeling. Understanding the function of KIM-1 will provide important insight into the role of this protein in injury and repair processes of the kidney, and may identify KIM-1 as an important therapeutic target not only for acute and chronic renal disease but also for malignant transformation of the epithelial cell.