Shh/Gli1-ABCG2通路调控肾脏边缘群细胞生物学行为参与缺血肾脏重建的机制

The kidney side population (SP) is a resident renal cell population with some stem/progenitor cell characteristics. Further study to illuminate the exact mechanism on regulating the biological behavior of kidney SP cells under hypoxic/ischemic condition may contribute to confirming the therapeutic target for regeneration of acute kidney injury. We previously demonstrated that ABCG2 is the functional protein of SP cells, and SDF-1α-CXCR4/CXCR7 axis regulates the expression of Shh/Gli1 and ABCG2 under hypoxic condition. It has been approved that Shh/Gli1, which is a downstream target of SDF-1α/CXCR4 axis, can regulate and activate many genes including ABCG2. Therefore, we presume that SDF-1α-CXCR4/CXCR7 axis induces the activation of Shh/Gli1, which regulates the transcription of ABCG2 and biological behavior in SP cells. In this study, we will first evaluate the role of ABCG2 on the biological behavior and function in SP cells. Secondly, by upregulating or downregulating the expression of correlative gene, the relevance of SDF-1α-CXCR4/CXCR7 axis-Shh/Gli1-ABCG2 pathway and its role on biological behavior of SP cells will be confirmed. Thirdly, the Gli1-binding site on the ABCG2 promoter will be evaluated using luciferase reporter, site-directed mutagenesis, chromatin immunoprecipitation assay (ChIP) and electrophoresis mobility shift assay (EMSA). Finally, using wild type and ABCG2 gene knockout mice to establish the renal I/R injury model, we will perform in vivo experiment to investigate the effect of Shh/Gli1 on phenotype and renal protective function of kidney SP cells. This study will provide the theoretic and experimental foundations for manipulating kidney SP cells to regenerate injured kidney tissue.

肾脏边缘群（SP）细胞是潜在的肾脏常驻干细胞群，探索其在缺血/缺氧下生物学行为的调控机制，有望揭示急性肾损伤再生医学疗法的新靶点。我们前期研究证实ABCG2是SP细胞的功能蛋白，SDF-1α-CXCR4/CXCR7轴调控了低氧下Shh/Gli1和ABCG2的表达。基于ABCG2是Gli1靶基因的文献回顾，我们推测低氧下SDF-1α-CXCR4/CXCR7轴诱导SP细胞Shh/Gli1活化，进而调控ABCG2转录和生物学行为，参与肾脏重建。本研究通过基因转染上调和下调相关通路基因，评价SDF-1α-CXCR4/CXCR7轴-Shh/Gli1-ABCG2通路的关联性以及在SP细胞生物学行为方面的作用；验证ABCG2启动子的Gli1结合位点；利用基因敲除小鼠建立肾脏I/R模型，以Shh/Gli1分子作为调控SP细胞生物学行为的靶点进行体内试验，从而为临床调控SP细胞参与缺血肾脏重建提供实验依据。

背景和目的：肾脏边缘群（side population，SP，也叫侧群）细胞已经被证实是富集肾脏干/祖细胞的细胞群，体外培养具有多向分化潜能，体内移植可以促进受损肾脏的修复。因此，本课题进一步研究其基本表型乳腺癌耐药蛋白(BCRP1/ABCG2)及潜在的上游信号通路在肾脏SP细胞抵抗缺血/缺氧损伤中的修复机制。.内容和结果：1）ABCG2特异性表达在肾脏SP细胞。2）ABCG2关联于氧糖剥夺（OGD）诱导的肾脏SP细胞存活能力、增殖和旁分泌作用。3）低氧增加了sonic hedgehog（Shh）在肾脏的体内和体外表达；而虎杖苷诱导了Shh表达的进一步增加。缺血和虎杖苷均诱导了肾脏细胞Gli1的核易位。4）阻断Shh信号，削弱了虎杖苷的抗凋亡和抗氧化应激效果。5）Shh信号参与了虎杖苷保护肾小管上皮细胞（RTECs）抵抗OGD诱导的凋亡和氧化应激；6）缺血后肾脏和模拟I/R后的SP细胞中SDF-1/CXCR4的表达增加；SDF-1/CXCR4轴保护肾脏SP细胞避免SI/R损伤；7）SDF-1/CXCR4轴负责着SP细胞趋化性和旁分泌作用，关联于SP细胞向SI/R受损RTECs的移动。8）SDF-1通过Shh/Gli1信号调控SI/R处理下肾脏SP细胞ABCG2的表达。9）阻断CXCR4信号削弱了SP细胞在肾脏I/R损伤中的效果。.科学意义：SDF-1/CXCR4-Shh/Gli1-ABCG2通路在肾脏SP细胞抵抗缺血/缺氧损伤中发挥了重要作用。Shh/Gli1通路可能是I/R损伤后肾脏重建的潜在治疗靶点，尚有待进一步深入研究。