Objective: To observe the relationship of kidney injury molecule-1 ( KIM-1 ) and hypoxia-inducible factor-1α (HIF-1α) expression in the hypoxia/reoxygenation phases of HK-2 cell line, and to investigate the mechanism of the regulation of KIM-1 by HIF-1α.Methodology:The HK-2 cells were collected in different phases of hypoxia and reoxygenation to detect the expression of KIM-1 and HIF-1α by RT-PCR and Western-Blot, and the effects of agonist (Cocl2 ) /inhibitor (Rapamycin) of HIF-1α on the expression of KIM-1 were observed.Further more, the combination of the promoter region of KIM-1 and HIF-1α was validated by EMSA and ChIP.Results:The mRNA/protein expression of KIM-1 was correlated to that of HIF-1α in hypoxia/reoxygenation phases of HK-2 cell line.The agonist ( Cocl2 ) and inhibitor (Rapamycin) of HIF-1α could respectively up-regulate and down-regulate the expression of KIM-1 expression,which was dose dependent.EMSA demonstrated that the designed Double-Stranded Olignnueleotide Probes based on the HRE combining site of the promoter region of KIM-1 could combined with HIF-1α, and this kind of combination was ultimately validated through ChIP.Conclusion: The expression of KIM-1 could be regulated by HIF-1α in hypoxic proximal tubular injury, which illustrated that KIM-1 was potentially a downstream molecule of HIF-1 involved in processes of injury and repair of the renal tubular epithelium.%目的:观察肾损伤分子-1(KIM-1)及缺氧诱导因子1α(HIF-1α)在人近端小管上皮细胞(HK-2)缺氧复氧过程中的表达及相互关系,探讨HIF-1α对KIM-1表达的调控作用及机制.方法:收集缺氧、缺氧复氧后不同时段培养的HK-2细胞,用RT-PCR和Western-Blot检测KIM-1及HIF-1α表达,同时观察HIF激动剂(CoCl2)及抑制剂(雷帕霉素)对KIM-1表达的影响;应用凝胶迁移实验(EMSA)和染色质免疫沉淀实验(ChIP)验证KIM-1启动子区和HIF-1结合情况.结果:HK-2细胞缺氧复氧过程中KIM-1和HIF-1在mRNA水平和蛋白水平表达相关,HIF-1激动剂CoCl2或抑制剂雷帕霉素可上调或下调KIM-1表达,并呈剂量依赖性;EMSA表明HIF-1α可以和KIM-1启动子区HRE结合位点为模板所设计的双链寡核苷酸探针特异性结合,而且这种特异性结合受HIF-1α调节,同时ChIP实验证实了KIM-1启动子区和HIF-1α的结合.结论:KIM-1在近端肾小管上皮细胞缺氧损伤时的表达受HIF-1α调控,KIM-1可能作为HIF-1的下游分子参与肾损伤及修复过程.
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