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目的观察白蛋白对人近端肾小管上皮细胞株(HK-2)血管紧张素转换酶2(ACE2)mRNA和蛋白表达的影响,探讨蛋白尿激活肾脏局部RAS的机制。方法采用不同浓度的牛血清白蛋白(BSA)作用HK-2细胞不同时间。以实时RT-PCR和Western印迹检测ACE2 mRNA和蛋白的表达。采用放射免疫法检测细胞上清液中血管紧张素Ⅱ(ATⅡ)含量。采用激光共聚焦显微镜观察ACE2蛋白的表达。结果与对照组(相对表达量为0)相比,BSA使ACE2 mRNA表达显著减少(2.5mg/ml:—1.05±0.12;5mg/ml:—1.30±0.11;10mg/ml:—2.54±0.44;P均<0.05)。同时,BSA使ACE2蛋白表达显著降低(对照组:0.90±0.10;2.5mg/ml:0.66±0.09;5mg/ml:0.50±0.07;10mg/ml:0.35±0.05;P均<0.05)。其中BSA(10mg/m1)使ACE2 mRNA及蛋白表达减少呈时间依赖性(P均<0.05)。与对照组相比,不同浓度BSA组的细胞上清液中的ATⅡ均显著升高(P均<0.05)。激光共聚焦显微镜可见ACE2主要分布于细胞膜。结论BSA可显著抑制HK-2细胞ACE2 mRNA和蛋白的表达,此作用所导致的近曲小管间质液ATⅡ浓度升高可能与间质纤维化相关。
Objective To investigate the effect of albumin on mRNA and protein expression of angiotensin-converting enzyme 2 (ACE2) in human proximal tubular epithelial cell line (HK-2) and to explore the mechanism of proteinuria in activating renal local RAS. Methods Different concentrations of bovine serum albumin (BSA) were used to effect HK-2 cells at different times. ACE2 mRNA and protein expression was detected by real-time RT-PCR and Western blotting. Radioimmunoassay was used to detect the content of angiotensin Ⅱ (AT Ⅱ) in the cell supernatant. The expression of ACE2 protein was observed by laser confocal microscopy. Results BSA significantly reduced ACE2 mRNA expression (2.5mg / ml: -1.05 ± 0.12; 5mg / ml: -1.30 ± 0.11; 10mg / ml: -2.54 ± 0.44, compared with the control group (relative expression 0) P <0.05). Meanwhile, BSA significantly reduced ACE2 protein expression (control group: 0.90 ± 0.10; 2.5mg / ml: 0.66 ± 0.09; 5mg / ml: 0.50 ± 0.07; 10mg / ml: 0.35 ± 0.05; all P <0.05). BSA (10mg / ml) reduced the expression of ACE2 mRNA and protein in a time-dependent manner (all P <0.05). Compared with the control group, ATⅡ in the supernatant of BSA group was significantly increased (all P <0.05). Confocal laser microscopy showed ACE2 mainly distributed in the cell membrane. Conclusion BSA can significantly inhibit the expression of ACE2 mRNA and protein in HK-2 cells. The increased concentration of proximal tubulointerstitial fluid ATⅡ may be related to interstitial fibrosis.