转录因子Six1调控肾源性分子表达促进肾脏损伤修复

Transcription factor Six1 regulates expression of nephrogenic molecules to promote repair of kidney injury

目的 探讨急性肾损伤(acute kidney injury,AKI)后肾源性转录因子正弦眼同源异型盒1(sine oculis homeobox 1,Six1)调控损伤修复的作用机制。方法 将C57BL/6J小鼠进行肾脏缺血再灌注损伤(ischemia reperfusion injury,IRI)实验,并于造模后第1、2和3天收集血清样品与肾脏组织;检测血肌酐、血尿素氮水平及肾脏HE染色;检测肾源性分子(Six1、Pax2等)、细胞增殖/迁移基因(Cyclin A1、MMP9等)表达及分布。在Six1过表达/基因敲除及氯化钴诱导损伤等细胞模型中,检测凋亡通路(Bcl2、Caspase3等)及细胞增殖等分子的表达。此外,利用腺相关病毒体内过表达Six1并建立IRI模型,检测Six1等相关分子的表达。结果 IR损伤后,Six1在上皮细胞中被显著激活;肾源性及细胞增殖/迁移分子(Pax2、Cyclin A1、MMP9等)表达显著上调(P<0.05),并与Six1表达正相关,且增殖迁移分子(Ki67、MMP9)同样定位于小管上皮细胞。在Six1过表达细胞模型中,肾源性与细胞增殖/迁移分子(Pax2、Cyclin A1、MMP9等)表达水平也显著上调(P<0.05)。在过表达Six1肾脏中,肾源性及抗凋亡分子(Six2、Pax2、Bcl2等)表达上调,肾损伤分子(Kim1)表达减少;而在IR损伤第2天,过表达Six1肾脏中抗凋亡基因(Bcl2、Stat3)显著上调(P<0.05),损伤及凋亡分子(Kim1、Caspase3)显著下调(P<0.05)。在氯化钴诱导模型中,与对照正常细胞比较,细胞增殖率在Six1敲除细胞(TCMK1-Six1^(-/-))中显著下降(P<0.05),而在Six1过表达细胞(TCMK1-Six1)中升高(P<0.05);肾源性、抗凋亡通路及细胞增殖/迁移分子(Pax2、Bcl2、Cyclin A1、MMP9等)表达在TCMK1-Six1^(-/-)中减少,凋亡与肾损伤(Caspase3、Kim1)分子在TCMK1-Six1中显著下调(P<0.05)。结论 AKI后激活的Six1通过上调肾源性分子的表达,并诱导抗凋亡通路分子表达,促进肾小管上皮细胞的增殖/迁移,参与了IR诱导肾脏损伤的修复。

Objective To investigate the role and mechanism of sine oculis homeobox 1(Six1),a nephrogenic transcription factor,in regulating injury repair after acute kidney injury(AKI).Methods C57BL/6J mice were inflicted with renal ischemia reperfusion(IR) injury to establish AKI model,and then the serum samples and kidney tissues were collected at days 1,2,and 3 after modeling.Serum creatinine(Cr) and blood urea nitrogen(BUN) levels were measured and renal morphology was observed with HE staining.The expression and distribution of nephrogenic molecules(Six1 and Pax2,etc.) and cell proliferation/migration genes(Cyclin A1,MMP9,etc.) were detected with RT-PCR,Western blotting,and immunofluorescence assay and immunohistochemical assay.The expression of apoptosis pathway(Bcl2,Caspase3,etc.) and cell proliferation related molecules were evaluated in Six1 overexpression/knockout human epithelial cells(HK2) with CoCl_2-induced injury.Additionally,after the adeno-associated viruses carrying Six1 overexpression vector were used to overexpress the molecule in the mice,the expression of Six1 and other related molecules were detected after IR injury modeling.Results After renal IR injury,Six1 was significantly activated in epithelial cells,the expression of nephrogenic and cell proliferation/migration molecules(Pax2,Cyclin A1,MMP9,etc.) was obviously up-regulated(P<0.05),which was positively correlated with Six1 expression,while the proliferation/migration molecules(Ki67,MMP9) were also localized within the tubular epithelial cells.In cellular models of Six1 overexpression,the expression levels of nephrogenic and cell proliferation/migration molecules(Pax2,Cyclin A1,MMP9,etc.) were also notably up-regulated(P<0.05).In the mice with renal overexpression of Six1,the nephrogenic molecules as well as anti-apoptotic ones(Six2,Pax2,Bcl2,etc.) were up-regulated,while the expression of kidney injury-related molecule(Kim1) in kidneys was reduced in the renal tissues.While,in 2 d after IR injury,the anti-apoptotic gene(Bcl2,Stat3) was significantly up-regulated(P<0.05),and the apoptotic and injury molecules(Kim1,Caspase3) showed remarkable down-regulation(P<0.05) in the mice with renal Six1 overexpression.Furthermore,CoCl_2-inducion significantly decreased the cell proliferation rate in the Six1 knockout group(TCMK1-Six1~(-/-))(P<0.05) but increased the rate in the Six1 overexpression group(TCMK1-Six1) when compared to the control cells(P<0.05).And,the expression of nephrogenic,anti-apoptotic pathways and cell proliferation/migration molecules(Pax2,Bcl2,Cyclin A1,MMP9,etc.) were reduced in TCMK1-Six1~(-/-) group,and apoptosis and kidney injury molecules(Caspase3,Kim1) were significantly down-regulated in TCMK1-Six1 group(P<0.05).Conclusion Activation of Six1 after AKI can promote the proliferation/migration of renal tubular epithelial cells by up-regulating nephrogenic molecules and inducing anti-apoptotic pathway molecules,and then,participate in IR-induced renal injury repair.