慢性低氧状态对肾小球足细胞高通量钙激活 钾通道表达和功能的影响

Influence of chronic hypoxia on expression and function of high-flux Ca2+-activated K+ channel in cultured human glomerular podocytes

摘要:

目的 探讨慢性低氧对人肾小球足细胞高通量钙激活钾通道（BK 通道）表达和功能的影响，及 其在慢性肾脏病进展中的作用。方法 分化完全的条件永生性人类足细胞分别置于常氧（21% O2）和低氧（10% 或2% O2）条件下培养6 ～ 48 h，采用实时聚合酶链反应（real-time PCR）和Western blot 检测足细胞BK 通道α、 β3 及β4 亚基的表达；用电生理膜片钳技术记录全细胞模式下足细胞BK 通道功能的变化。结果 电生理研 究结果显示低氧环境（2% O2 24 h）中，足细胞BK 通道在+80 mV 电压刺激下的电流水平由（14.45±2.06） pS 下降至（4.78±1.12）pS，差异有统计学意义（P <0.05），且通道激活的时间常数由（4.59±1.67）增加至（25.16± 11.04）（P <0.05）；分子生物学研究提示在低氧条件（2% O2 24 h）下足细胞BK 通道的β4 亚基水平升高， 且表现为时间依赖性和剂量依赖性。结论 慢性低氧通过上调β4 亚基表达抑制BK 通道活性。

Abstract:

Objective To explore the influence of chronic hypoxia on expression and function of high-flux Ca2+- activated K+ channel (BK channel) in human glomerular podocytes, and to evaluate its roles in the progression of chronic kidney diseases (CKD). Methods A fully-differentiated and conditionally-immortalized human podocyte cell line was placed into the normoxic (21% O2) or hypoxic (2% O2 or 10% O2) environment in a duration of 6 to 48 h. The expressions of α, β3 and β4 subunits of BK channels were detected with real-time PCR and Western blot. The changes of BK channel function in podocytes under whole-cell model were examined by the patch clamp technique. Results Exposure of podocytes to 2% O2 for 24 h caused a significant reduction in currents at +80 mV from (14.45 ± 2.06) pS to (4.78 ± 1.12) pS (P < 0.05). Chronic hypoxia also caused a marked increase in the time constant for BK channel activation from (4.59 ± 1.67) to (25.16 ± 11.04) with significant difference (P < 0.05). The total expression of β4 subunit of BK channel significantly increased in a time- and dose-dependent manner. Conclusions Chronic hypoxia inhibits BK channel current by upregulating β4 subunit expression.