CaMKⅡ/NFAT通路激活可促进AKI小鼠肾小管上皮细胞凋亡

Activation of CaMKⅡ/NFAT signaling pathway promotes renal tubular epithelial cell apoptosis in AKI mice

目的:研究钙/钙调蛋白依赖性蛋白激酶Ⅱ(calcium/calmodulin-dependent protein kinaseⅡ,CaMKⅡ)和活化T细胞核因子(nuclear factor of activated T cells,NFAT)是否参与急性肾损伤(acute kidney injury,AKI)中肾小管上皮细胞(renal tubular epithelial cells,RTEC)凋亡并探讨其机制。方法:阿霉素(adriamycin,ADR)干预人近端肾小管上皮细胞HK-2建立RTEC凋亡模型,小干扰RNA(small interfering RNA,siRNA)沉默NFAT基因,流式细胞术测细胞凋亡率。ADR干预6~8周龄雌性BALB/c小鼠建立AKI模型,将小鼠分为3组:正常对照组、ADR组和ADR+CaMKⅡ抑制剂KN-93干预组,每组5只。酶联免疫吸附实验检测小鼠血清肌酐浓度。Western blot检测体内外Bax、Bcl-2、p-CaMKⅡ、CaMKⅡ及核NFAT的蛋白水平。结果:与正常对照组相比,ADR干预下,HK-2细胞凋亡率显著升高(P<0.01),小鼠血清肌酐和Bax蛋白表达显著升高,而Bcl-2蛋白表达显著下降(P<0.05)。ADR干预下,HK-2细胞和小鼠肾组织中CaMKⅡ活性(p-CaMKⅡ/t-CaMKⅡ)与核蛋白NFAT表达皆显著升高(P<0.01)。ADR干预的HK-2细胞和小鼠中抑制CaMKⅡ活性后,HK-2细胞凋亡率显著降低,小鼠血清肌酐和Bax蛋白表达显著回降,而Bcl-2蛋白表达显著回升,核蛋白NFAT表达显著回降(P<0.05)。此外,用钙调蛋白激活CaMKⅡ后HK-2细胞凋亡率显著升高(P<0.05);激活CaMKⅡ同时予siRNA沉默NFAT,HK-2细胞凋亡率显著降低(P<0.05)。结论:CaMKⅡ/NFAT通路激活可促进AKI小鼠肾小管上皮细胞凋亡。

AIM:To verify whether calcium/calmodulin-dependent protein kinaseⅡ(CaMKⅡ)and nuclear factor of activated T cells(NFAT)are involved in the apoptosis of renal tubular epithelial cells(RTEC)in acute kidney injury(AKI).METHODS:The RTEC apoptosis model was induced by adriamycin(ADR)in cultured human kidney proximal tubule epithelial cell line HK-2.Apoptosis rate was detected by flow cytometry.The AKI model was induced by ADR in 6 to 8-week-old female BALB/c mice.The mice were divided into 3 groups:normal control group,ADR group,and ADR+CaMKⅡinhibitor KN-93 group,with 5 mice in each group.Serum creatinine was detected by enzyme-linked immunosorbent assay.The protein levels of Bax,Bcl-2,p-CaMKⅡ,CaMKⅡand nuclear NFAT were detected by Western blot in vitro and in vivo.RESULTS:Comparing with normal control group,the apoptosis rate of ADR-injured HK-2 cells was significantly increased(P<0.01),and serum creatinine and renal Bax protein expression were significantly increased,while the protein expression of Bcl-2 was decreased in ADR-treated mice(P<0.05).Both CaMKⅡactivity(pCaMKⅡ/t-CaMKⅡ)and the nuclear protein expression of NFAT were significantly increased in ADR-injured HK-2 cells and kidney tissue cells of ADR-treated mice(P<0.01).Apoptosis,serum creatinine,and the protein expression of Bax,Bcl-2 and nuclear NFAT were significantly ameliorated by inhibition of CaMKⅡactivity in ADR-injured HK-2 cells and mice(P<0.05).Furthermore,apoptosis rate of HK-2 cells was significantly inceased after treatment with CaMKⅡactivator calmodulin(P<0.05),but it was significantly decreased by NFAT gene silencing with siRNA(P<0.05).CONCLUSION:Activation of CaMKⅡ/NFAT signaling pathway could promote RTEC apoptosis in AKI mice.