钙调蛋白S100A16诱导细胞骨架重构参与肾脏上皮细胞间充质转分化的分子机制

Renal fibrosis, particularly tubulointerstitial fibrosis, is the common final outcome of almost all progressive chronic kidney diseases. Renal tubulointerstitial fibrosis is also a reliable predictor of prognosis and a major determinant of renal insufficiency. Epithelial-mesenchymal transition (EMT), a process by which fully differentiated epithelial cells undergo transition to a fibroblast phenotype, has emerged as an important pathway leading to generation of matrix-producing fibroblasts and myofibroblasts in renal fibrosis. A key feature of EMT is reorganization of the cytoskeleton. S100A16, a Ca+ binding protein, is a new member of S100 protein family. However, the study of S100A16 in renal tubulointerstitial fibrosis is very rare. The preliminary data from our proposed project has shown that S100A16 was a key mediator of EMT, and contributed the progression of renal tubulointerstitial fibrosis in mice model as well as human kidney disease. Moreover, we found that there were many cytoskeleton and cytoskeletal associated proteins, such as Cofilin, binding with S100A16. Based on these preliminary data, we’ll further study the molecular mechanism of S100A16 regulation of cytoskeletal reorganization during EMT in renal tubular cells and transgenic/knock out mice model. We will demonstrated how changes in gene expression patterns of cytoskeletal associated proteins during S100A16-induced EMT, and how changes in cytoskeletal architecture during EMT can regulate gene expression, thus further promoting EMT progression in renal tubulointerstitial fibrosis.

肾小管间质纤维化是慢性肾脏病发展的重要病理过程，在此过程中肾小管上皮细胞间充质转分化(EMT)是发病的关键环节。细胞骨架蛋白表达与重组是EMT的重要特征。钙调蛋白S100A16是新发现的S100家族成员，其病生理功能不详。我们的前期研究表明：S100A16的过表达加重肾小管间质纤维化，促进肾小管上皮细胞EMT；质谱结果提示S100A16与大量的细胞骨架蛋白和肌动蛋白调控相关的蛋白存在相互作用(如cofilin)，对相关细胞骨架蛋白的表达和功能产生影响。我们假设各种肾脏损伤因素上调肾小管上皮中S100A16的表达，进而增强其与肌动蛋白调节蛋白的结合能力，诱发肾脏上皮细胞骨架蛋白重构和EMT的发生，参与肾小管间质纤维化的发生发展。本课题将利用临床标本、动物及分子生物学实验、蛋白质功能筛选等技术进一步证实课题假设，揭示S100A16通过诱导细胞骨架蛋白重构引起肾脏上皮细胞间充质转分化的分子机制。

肾小管间质纤维化（renal tubulointerstitial fibrosis）是慢性肾脏病发展的重要病理过程，在此过程中肾小管上皮细胞间充质转分化（Epithelial intercellular transformation，EMT）是发病的关键环节。细胞骨架蛋白表达与重组是EMT的重要特征。钙结合蛋白S100A16是S100家族的新成员，在各器官和组织上广泛表达，但其病生理功能仍不清楚。因此，本课题将探究S100A16与肾小管间质纤维化之间的关系。本课题中我们选取不同程度慢性肾病患者的肾穿标本进行组织化学染色。结果显示：与正常肾组织相比，随着肾脏病变的加重S100A16表达增加，提示S100A16与肾脏病变程度相关。在S100A16转基因和敲除鼠上建立肾小管间质纤维化模型，我们发现S100A16高表达与肾小管间质纤维化的发生呈正相关，敲除S100A16则可以逆转肾小管间质纤维化。通过检测EMT的相关指标，我们发现S100A16通过促进EMT来加重肾小管间质纤维化。在体外实验中，用 TGF-β诱导人肾小管上皮细胞株（HK-2 cells）损伤。在TGF-β损伤的HK-2细胞中，过表达或敲除S100A16，再检测EMT的相关指标，其结果与小鼠实验一致。我们利用液相色谱/质谱分析了与S100A16相关的蛋白，骨架蛋白肌凝蛋白重链9（Myosin heavy chain 9，Myh9）是候选基因之一。通过免疫共沉淀和免疫荧光共定位确定S100A16与Myh9之间具有特异性相互作用。胞质钙离子浓度升高到一定程度时，钙离子与Myh9结合，使Myh9构型改变，致使肌凝蛋白分子构型变化移位，细胞骨架蛋白重组。使用毒胡萝卜素（thapsigargin，TG）降低胞内游离钙离子浓度抑制Myh9功能，或者通过抗体转染实验降低胞内Myh9的水平，均可逆转EMT。综上所述， S100A16通过提高胞内钙离子浓度，激活钙依赖性细胞骨架蛋白Myh9，促进EMT的发生，进而参与导致肾小管间质纤维化。本课题主要研究了S100A16促进EMT从而加重纤维化的分子机制，对临床上寻找预防和治疗肾小管间质纤维化的靶点具有重要的理论和指导意义。