醛固酮通过线粒体源性氧化应激促进表皮生长因子受体活化及肾小球系膜细胞增殖

目的 研究活性氧（ROS）在醛固酮（ALDO）诱导的表皮生长因子受体（EGFR）信号通路活化及系膜细胞增殖中的作用并探讨ROS的来源。 方法 体外培养人肾小球系膜细胞，应用3H-胸腺嘧啶（3H-TdR）掺入法和细胞计数测定系膜细胞增殖；荧光探针2，7-二氯二氢荧光素乙酰乙酸（DCFDA）检测细胞内ROS 的产生；Western印迹法检测EGFR活化。 结果 ALDO可显著促进肾小球系膜细胞增殖，应用100 nmol/L ALDO刺激系膜细胞24 h后，3H-TdR掺入量及细胞数分别是基础状态下的2.63倍和2.15倍。盐皮质激素受体（MR）阻断剂依普利酮（EPLE）几乎完全阻断ALDO诱导的系膜细胞增殖（P < 0.01），而糖皮质激素受体（GR）阻断剂RU-486对ALDO诱导的细胞增殖无显著抑制作用。ALDO明显促进肾小球系膜细胞ROS产生，100 nmol/L ALDO刺激60 min，系膜细胞内ROS释放是对照组的2.14倍，EPLE显著抑制ALDO诱导的系膜细胞ROS产生（P < 0.01）。线粒体复合体 I抑制剂鱼藤酮（ROT）几乎完全阻断ALDO诱导的ROS产生（P < 0.01），NADPH 氧化酶抑制剂夹竹桃麻素（apocynin）和二联苯碘（DPI）对ALDO诱导ROS产生的抑制率为30%~35%（P < 0.05），而黄嘌呤氧化酶抑制剂别嘌醇、环氧化酶抑制剂吲哚美辛、脂氧化酶抑制剂去甲二氢化愈创木酸、细胞色素P450氧化酶抑制剂酮康唑以及一氧化氮合成酶抑制剂N-硝基-L-精氨酸甲酯对ALDO诱导的ROS产生均无明显影响。乙酰半胱氨酸（NAC）和ROT对ALDO诱导系膜细胞增殖的抑制率为75%~80%，apocynin和DPI的抑制作用仅为25%~30%。ALDO可显著活化系膜细胞EGFR，ALDO刺激60 min，EGFR活性是对照组的4.95倍，EPLE和NAC几乎完全阻断ALDO诱导的EGFR磷酸化（P < 0.01），而NAC和EGFR拮抗剂AG1478则阻断ALDO诱导的系膜细胞增殖（P < 0.01）。 结论 ALDO通过氧化应激依赖的EGFR磷酸化促进肾小球系膜细胞增殖，ALDO诱导的系膜细胞氧化应激主要来源于线粒体。

Mitochondrial derived reactive oxygen species mediates aldosterone-induced epidermal growth factor receptor activation and mesangial cell proliferation

Objective To detect the signaling pathways involved in aldosterone (ALDO)-induced mesangial cell (MC) proliferation. Methods The incorporation of 3H-thymidine (3H-TdR) and cell count were used as the measure of mesangial cell (MC) proliferation. Reactive oxygen species (ROS) production was determined by DCFDA fluorescence. Epidermal growth factor receptor (EGFR) activation was assayed by Western blotting. Results ALDO induced MC proliferation. When incubation with 100 nmol/L ALDO for 24 h, the 3H-TdR incorporation and cell number increased by 2.63- and 2.15-fold, respectively. Mineralocorticoid receptor (MR) antagonist EPLE almost completely blocked ALDO-induced MC proliferation (P<0.01), however, glucocorticoid receptor (GR) antagonist RU-486 had no effect on MC proliferation. ALDO increased intracellular ROS production in cultured human MCs. When incubation with ALDO (100 nmol/L) for 60 min, ROS production increased by 2.14-fold. ALDO-induced ROS generation was completely blocked by EPLE as well as mitochondrial complex I inhibitor rotenone (P<0.01), NADPH oxidase inhibitors diphenyleneiodonium sulfate (DPI) and apocynin inhibited ALDO-induced ROS production by 30% to 35% (P<0.05). In contrast, inhibitors of other oxidant-producing enzymes, including allopurinol, indomethacin, nordihydroguiaretic acid, ketoconazole and G-nitro-L-arginine methyl ester (L-NAME) had no effect on ALDO-induced ROS production. Antioxidant N-acetyl-L-cysteine (NAC) and ROT inhibited ALDO-induced MC proliferation by 75% to 80%, whereas the inhibition of NADPH oxidase inhibitor apocynin and DPI on ALDO-induced MC proliferation was 25% to 30%. ALDO induced EGFR transactivation. When incubation with 100 nmol/L ALDO for 60 min, EGFR phosphorylation was increased by 4.95-fold, which was completely inhibited by EPLE and antioxidant NAC (P<0.01). NAC and EGFR antagonist AG1478 significantly blocked ALDO-induced MC proliferation (P<0.01). Conclusions ALDO-induced MC proliferation is mediated by ROS-dependent EGFR transactivation. ALDO-stimulated ROS is mainly generated by mitochondria.