草酸和草酸钙结晶损伤肾小管上皮细胞的差异蛋白质组学研究

目的 分析并鉴定草酸和一水草酸钙(COM)结晶损伤人肾小管上皮细胞(HK-2)后细胞蛋白质谱的表达变化,探讨肾小管上皮细胞受损在肾结石形成中的可能作用.方法 体外培养正常HK-2细胞至90%融合后,换无血清培养基,随机分为2组.实验组培养基内加入2 mmol/L草酸+200 mg/L COM结晶,37 ℃孵育.分别抽提2组细胞的总蛋白,双向凝胶电泳结合液相色谱-电喷雾离子阱质谱(LC-ESI-MS/MS)技术对2组中差异表达的蛋白质进行分离和鉴定.蛋白印迹法对鉴定出的蛋白进行验证.结果 成功建立细胞总蛋白的双向凝胶电泳图谱,经软件分析和质谱鉴定出差异蛋白质12个:FK506结合蛋白4、α-烯醇酶、M2型丙酮酸激酶、ATP合成酶α亚单位、3′,5′-二磷酸核苷酸酶1、核磷蛋白2、L-乳酸脱氢酶B、芽胞发芽蛋白3、Cofilin-1、Fascin、40S核糖体蛋白S17和胞液氨基肽酶1.差异蛋白涉及细胞能量代谢、细胞增殖、凋亡、钙离子通道活性调控、细胞运动及信号转导等多种生理活动.蛋白印迹法检测示HK-2细胞损伤后ENO1蛋白表达上调,而Cofilin-1表达下调,证实双向凝胶电泳和质谱分析可靠.结论 高浓度草酸和COM结晶可使正常人HK-2细胞蛋白表达发生改变,这些差异表达蛋白既可起到细胞的自我保护作用,又可能通过相应途径在肾结石的形成中起重要作用.

Abstract：

Objective To analyze and identify the differentially expressed proteins in human renal tubular epithelial cells (HK-2) after injury caused by oxalic acid and calcium oxalate monohydrate (COM) crystal, and to explore the potential role of renal tubular cell injury in kidney stone formation.Methods Normal HK-2 cells were cultured in vitro and the culture medium was changed with serum-free medium after cell growth to confluence. Oxalic acid and COM crystals (final concentration at 2 mmol/L and 200 mg/L, respectively) were added in the experimental group. Cells in both groups were then incubated at 37 ℃ for 12 h. The extracted proteins from both groups were separated by two dimensional electrophoresis followed by analysis, and the differentially expressed proteins were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Two identified proteins were then verified by western blot. Results Reproducible two dimensional gel images of the proteins from both groups were successfully obtained. By using LC-ESI-MS/MS, 12 proteins: FK506-binding protein 4, isoform alpha-enolase of alpha-enolase, isoform M1 of pyruvate kinase isozymes M1/M2, ATP synthase subunit alpha, isoform 1 of 3′(2′), 5′-bisphosphate nucleotidase 1, isoform 2 of nucleophosmin, L-lactate dehydrogenase B chain, Budding uninhibited by benzimidazoles 3, Cofilin-1, Fascin, pyIsoform 1 of cytosol aminopeptidase, were identified. The deferentially expressed proteins were related to cellular processes including energy metabolism, cell multiplication, apoptosis, Ca2+ channel activity regulation, cell movement and signal transduction. Western blot verified that higher ENO1 but lower Cofilin-1 expressed in HK-2 cells after the injury. Conclusions High level oxalic acid and COM crystals can cause protein expression profile changes in normal human HK-2 cells. The changes of protein expression may not only protect HK-2 cells from being injured, but also be related to kidney stone formation.

Comparative proteomic analysis of renal tubular epithelial cell injury caused by oxalic acid and calcium oxalate monohydrate

Objective To analyze and identify the differentially expressed proteins in human renal tubular epithelial cells (HK-2) after injury caused by oxalic acid and calcium oxalate monohydrate (COM) crystal, and to explore the potential role of renal tubular cell injury in kidney stone formation.Methods Normal HK-2 cells were cultured in vitro and the culture medium was changed with serum-free medium after cell growth to confluence. Oxalic acid and COM crystals (final concentration at 2 mmol/L and 200 mg/L, respectively) were added in the experimental group. Cells in both groups were then incubated at 37 ℃ for 12 h. The extracted proteins from both groups were separated by two dimensional electrophoresis followed by analysis, and the differentially expressed proteins were identified by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Two identified proteins were then verified by western blot. Results Reproducible two dimensional gel images of the proteins from both groups were successfully obtained. By using LC-ESI-MS/MS, 12 proteins: FK506-binding protein 4, isoform alpha-enolase of alpha-enolase, isoform M1 of pyruvate kinase isozymes M1/M2, ATP synthase subunit alpha, isoform 1 of 3′(2′), 5′-bisphosphate nucleotidase 1, isoform 2 of nucleophosmin, L-lactate dehydrogenase B chain, Budding uninhibited by benzimidazoles 3, Cofilin-1, Fascin, pyIsoform 1 of cytosol aminopeptidase, were identified. The deferentially expressed proteins were related to cellular processes including energy metabolism, cell multiplication, apoptosis, Ca2+ channel activity regulation, cell movement and signal transduction. Western blot verified that higher ENO1 but lower Cofilin-1 expressed in HK-2 cells after the injury. Conclusions High level oxalic acid and COM crystals can cause protein expression profile changes in normal human HK-2 cells. The changes of protein expression may not only protect HK-2 cells from being injured, but also be related to kidney stone formation.