Src蛋白在急性肾损伤后肾小管上皮细胞去分化中的调控作用

The prognosis of acute kidney injury (AKI) is closely associated with renal tubular cell repair and regeneration. It has been recognized that dedifferentiation of renal tubular cells is a necessary prerequisite for other regenerative responses (i.e. proliferation). However, the regulatory mechanism of renal epithelial cell dedifferentiation after AKI remains unclear. Our previous studies revealed that following oxidant injury (1) epidermal growth factor receptor (EGFR) and Src, a non-receptor tyrosine kinase, are activated in renal tubular epithelial cells, (2)Src mediates EGFR activation and (3)EGFR activation is required for renal epithelial dedifferentiation. These studies suggest that Src-mediated EGFR activation may play an important role in renal epithelial cell dedifferentiation. By transfecting siRNA and dominant negative mutants, and using immunoprecipitation technique, we will further determine the role of Src and amphiregulin(AR), the EGFR ligand, in renal epithelial cell dedifferentiation. We will also explore the mechanism by which Src induces EGFR activation in renal epithelial cells following oxidant injury. Furthermore, we will assess the role of the Src-AR-EGFR pathway in renal epithelial cell dedifferentiation, proliferation and renal function recovery in a mouse model of ischemia/reperfusion-induced AKI by pharmacological and genetic blockade of Src and EGFR and siRNA-mediated sliencing of AR. Successful completion of these studies will lay the groundwork for elucidation of the regulatory mechanism of renal tubular cell dedifferentiation and will establish the molecular basis for targeting acceleration of renal regeneration after AKI.

急性肾损伤 (AKI)的预后与肾小管细胞的自身修复和再生密切相关。已知AKI后肾小管上皮细胞去分化是其它再生反应（如增生）的前提。但分子调控机制不明。课题组前期研究结果表明氧化损伤后的肾小管上皮细胞中Src蛋白和表皮生长因子受体（EGFR）的活性増加。Src介导EGFR激活，EGFR活化与肾上皮细胞去分化有关。提示Src介导的EGFR激活在急性损伤后肾小管上皮细胞去分化中起重要作用。本项目拟利用siRNA沉默，基因突变，免疫沉淀等技术，进一步探讨Src和EGFR的配体，双调蛋白（AR），在肾小管上皮细胞损伤后去分化中的调控作用，明确Src活化EGFR的分子机制；并通过应用药物抑制剂，基因敲除或缺陷鼠评估Src-AR-EGFR通路在小鼠急性肾缺血/再灌注损伤后的肾小管上皮细胞去分化和肾功能恢复中的作用。为阐明AKI后肾小管上皮细胞去分化的调控机制奠定基础，为促进肾再生的靶向治疗提供理论依据。

急性肾损伤 (AKI)的预后与肾小管细胞的自身修复和再生密切相关。已知AKI后肾小管上皮细胞去分化是其它再生反应（如增生）的前提。但分子调控机制不明。课题组前期研究结果表明氧化损伤后的肾小管上皮细胞中Src蛋白和表皮生长因子受体（EGFR）的活性増加。Src介导EGFR激活，EGFR活化与肾上皮细胞去分化有关。提示Src介导的EGFR激活在急性损伤后肾小管上皮细胞去分化和肾损伤中起作用。在本研究中， 我们首先利用小鼠急性肾缺血/再灌注模型探讨了Src在肾上皮细胞死亡和急性肾损伤中的作用。发现应用 PP1抑制Src明显改善肾功能， 减轻肾小管上皮细胞的损伤标志,NGAL (neutrophil gelatinase-associated lipocalin)的表达和细胞凋亡。另外，抑制Src也防止细胞联接蛋白，E-cadherin, ZO-1, and claudins-1/-4 的表达和降低细胞金属蛋白酶-MMP2和MMP-9的活化， 以及促细胞凋亡蛋白-extracellular signal–regulated kinases1/2和促炎症因子- signal transducer and activator of transcription-3, and nuclear factor-κB的磷酸化，同时减轻肾脏巨噬细胞的侵润 。这些研究结果表明，尽管Src 在体外介导肾上皮细胞的去分化反应，但是由于其在体内的激活导致细胞联接蛋白表达减少和其他致病因子的活化，最终Src 激酶在急性肾损伤中发挥致病作用。 由于严重的肾损伤往往导致肾小管结构不全修复和继发慢性肾脏纤维化， 故我们尽一步研究了Src在肾脏纤维化中的作用。发现Src在培养的成纤维细胞中和单侧输尿管结扎（UUO）致肾脏纤维化模型小鼠的肾脏中表达明显增加。应用PP1阻断 Src抑制体内体外成纤维细胞的活化，和在UUO模型中细胞外基质的沉积。此外，在体外实验中，PP1 也有效抑制促纤维化因子TGF-beta引起的成纤维细胞的活化， 并降低TGF-beta 信号通路的激活，上皮生长因子受体（EGFR）和血小板生长衍生因子受体beta（PDGFR-beta）的磷酸化水平，以及其下游信号分子，STAT3， NF-kappB的激活。 此外， Src抑制也使细胞周期蛋白的标志物组蛋白H3磷酸化位点10 表达减低。 这些