Src Family Kinases and Control of Epithelial Cell Paracellular Permeability

描述（由申请人提供）：溶质和水通过上皮细胞（跨细胞）或细胞之间通过紧密连接（旁细胞）穿过上皮层。维持细胞旁通透性屏障对肾上皮的正常功能至关重要。在肾缺血/再灌注（I/R）损伤时，细胞旁通透性屏障被破坏，导致肾功能丧失。Src家族激酶（SFK）活性与介导I/ r诱导的屏障功能丧失有关，但这是有争议的，单个SFK的作用尚不清楚。基于先前的研究和我们的初步结果，我们假设肾上皮细胞的细胞旁通透性屏障是通过SFK c-yes与紧密连接蛋白occludin的相互作用维持的。肾I/R损伤，部分由局部H2O2增加介导，导致c-yes与occludin分离，c-src（另一种SFK）与occludin关联。这导致封闭蛋白在紧密连接中的停留时间减少，从而增加细胞旁通透性。第一系列实验将验证c-yes活性降低和c-src活性增加肾上皮细胞旁通透性的假设。实验将采用遗传技术来操纵这两种sfk的活性，并将监测细胞旁通透性的两个组成部分，即孔隙途径（高容量，小尺寸-通过测量表皮传导阻力（TER）来监测）和泄漏途径（低容量，大尺寸-通过测量钙黄蛋白和荧光素-葡聚糖4000的通量来监测）。第二系列实验将确定H2O2对occludin与c-src和c-yes相互作用的影响。实验将利用基因技术来操纵SFK活性和occludin含量。Occludin与c-src和c-yes的相互作用以及紧密连接结构的定位将通过共聚焦显微镜进行监测。第三系列实验将直接验证H2O2处理导致occludin在紧密连接结构中停留时间减少的假设，并且这种减少是由c-src活性介导的。这些实验将使用活细胞的共聚焦显微成像来监测occludin蛋白的动态行为。第四系列实验将确定在大鼠肾I/R损伤模型中是否存在类似的机制。单个SFK内容物将使用一种独特的慢病毒递送系统来操纵目标构建物到近端小管段。I/R损伤的严重程度将通过测量反映肾功能、肾小球滤过率（血清/尿肌酐比）和近端小管细胞结构完整性、Na+-K+- atp酶极性的参数来监测。这些研究将明确肾I/R诱导的肾上皮细胞旁通透性屏障丧失的机制，并将为开发治疗性干预措施以减少肾I/R损伤提供依据。

DESCRIPTION (provided by applicant): Solutes and water move across epithelial layers either through the epithelial cells (transcellular) or between cells through the tight junction (paracellular). Maintenance of the paracellular permeability barrier is essential for normal function of renal epithelia. During renal ischemia/reperfusion (I/R) injury, the paracellular permeability barrier is disrupted and contributes to loss of renal functional capacity. Src Family Kinase (SFK) activity has been implicated in mediating I/R-induced loss of barrier function but this is controversial and the roles of individual SFKs are unknown. Based on previous studies and our preliminary results, we hypothesize that renal epithelial cell paracellular permeability barrier is maintained through interaction of one SFK, c-yes, with the tight junction protein occludin. Renal I/R injury, mediated in part by localized increase in H2O2, causes dissociation of c-yes from occludin and association of c-src, another SFK, with occludin. This leads to a decreased occludin residency time in the tight junction which increases paracellular permeability. The first series of experiments will test the hypothesis that c-yes activity decrease and c-src activity increases renal epithelial cell paracellular permeability. Experiments will employ genetic techniques to manipulate the activities of these two SFKs and will monitor two components of paracellular permeability, the pore pathway (high capacity, small size - monitored by measuring TransEpithelial Resistance (TER)) and the leak pathway (low capacity, large size - monitored by measuring the flux of calcein and fluorescein-dextran 4000). The second series of experiments will define the effect of H2O2 on the interaction of occludin with c-src and c-yes. Experiments will utilize genetic techniques to manipulate SFK activities and occludin content. Occludin interaction with c-src and c-yes as well as localization to the tight junction structure will be monitored by confocal microscopy. The third series of experiments will test directly the hypothesis that H2O2 treatment produces a decrease in the residency time of occludin at the tight junction structure and that this decrease is mediated by c-src activity. Thes experiments will employ confocal microscopic imaging of live cells to monitor occludin protein dynamic behavior. The fourth series of experiments will determine if a similar mechanism is operative in a rat renal I/R injury model. Individual SFK contents will be manipulated using a unique lentivirus delivery system to target constructs to the proximal tubule segment. I/R injury severity will be monitored by measuring parameters reflecting renal functional capacity, glomerular filtration rate (serum/urinary creatinine ratio), and proximal tubule cell structural integrity, Na+-K+-ATPase polarity. These studies will define the mechanism underlying renal I/R-induced loss of renal epithelial cell paracellular permeability barrier and will provide the bais for development of therapeutic interventions to minimize renal I/R injury. PUBLIC HEALTH RELEVANCE: Movement of compounds across epithelial cell layers through the paracellular pathway (between the cells) is important for control of body solute and water balance. The experiments described in this proposal will define the molecular mechanism by which this pathway is disrupted during a pathogenic condition, renal ischemia/reperfusion injury, that contributes substantially to sickness and death in renal transplant patients and others.