激活SUR2B/Kir6.1亚型KATP通道对肾脏细胞损伤的干预作用及其机制*

doi:10.12047/j.cjap.6356.2022.110

摘要/Abstract

摘要： 目的: 探讨新型SUR2B/ Kir6.1亚型K_ATP通道开放剂埃他卡林对尿酸所致肾脏细胞(肾小球内皮、系膜和肾小管上皮细胞)损伤干预作用及其机制。方法: ①实验分组:对照组(0 mg/L尿酸损伤24 h);模型组(1 200 mg/L尿酸损伤24 h);埃他卡林预处理组(终浓度为0.01、0.1、1、10、100 μmol/L预孵育24 h +1 200 mg/L尿酸损伤24 h);K_ATP拮抗组(10 μmol/L 的格列苯脲孵育1 h 后,加入10 μmol/L埃他卡林共孵育24 h+1 200 mg/L尿酸损伤24 h)。②采用MTT法、流式细胞术检测细胞存活率;免疫荧光检测细胞Kir6.1、SUR2B的蛋白表达及细胞中NF-κB的核转位;Western blot检测细胞Kir6.1、SUR2B的蛋白表达;用荧光强度分析法检测单核细胞对内皮细胞的粘附;用酶联免疫吸附试验(ELISA)检测各组培养液中MCP-1抗原含量。结果: 1 200 mg/L尿酸作用于肾小球内皮、系膜和肾小管上皮细胞24 h后,与对照组相比,显著降低细胞存活率(P均＜0.01)。在肾小球内皮、系膜细胞损伤模型中,埃他卡林0.1、1、10、100 μmol/L预处理组与模型组相比,显著升高细胞存活率(P＜0.05, P＜0.01)。K_ATP拮抗剂组与对照组比,细胞存活率显著降低(P＜0.01),与模型组比无显著差异 (P＞0.05), 与埃他卡林10 μmol/L预处理组比有显著差异 (P＜0.05, P＜0.01)。在肾小管上皮细胞损伤模型中,埃他卡林10、100 μmol/L预处理组与模型组相比,显著升高细胞存活率 (P＜0.05)。K_ATP拮抗剂组与对照组比,细胞存活率显著降低(P＜ 0.01),与模型组比无显著差异(P＞0.05)。肾小球内皮、系膜和肾小管上皮细胞中,模型组与对照组相比,显著升高Kir6.1、SUR2B的蛋白表达 (P均＜0.05)。10 µmol/L埃他卡林预处理组与模型组相比,显著抑制上调Kir6.1、SUR2B蛋白的表达 (P均＜0.05)。K_ATP拮抗剂组Kir6.1、SUR2B的蛋白表达升高,与模型组比均无显著差异 (P＞0.05)。1 200 mg/L尿酸与肾小球内皮细胞孵育24 h后,与对照组相比,单核细胞对内皮细胞的黏附增多(P＜ 0.01)。埃他卡林10 µmol/L 预处理组与模型组相比,显著抑制单核细胞对内皮细胞的黏附(P＜0.05)。K_ATP拮抗剂组单核细胞对内皮细胞的黏附增多,与模型组无显著差异(P＞0.05)。1 200 mg/L尿酸刺激肾小球内皮细胞24 h后,与对照组相比,显著升高MCP-1分泌量 (P＜0.05)。埃他卡林10 µmol/L 预处理组与模型组比,显著降低MCP-1的分泌量(P＜0.05)。K_ATP拮抗剂组MCP-1的分泌量增多,与模型组比无显著差异 (P＞0.05)。肾小球内皮细胞受到尿酸刺激后,NF-κB从胞浆转位到胞核,当提前孵育ATP敏感性钾通道开放剂,再用尿酸刺激,可减少NF-κB转位到胞核。K_ATP拮抗剂可以逆转其作用。结论: 新型SUR2B/Kir6.1亚型K_ATP通道开放剂埃他卡林对尿酸所致肾脏细胞的损伤有明显的干预作用,其机制可能与激活细胞膜K_ATP通道蛋白有关。

关键词: SUR2B/Kir6.1亚型, ATP敏感性钾离子通道, 埃他卡林, 肾脏损伤, 高尿酸血症

Abstract: Objective: To investigate the interventional effects of a new SUR2B/Kir6.1-type K_ATP Channel opener iptakalim on injury renal cells (the renal glomerular endothelial, mesangial and tubular epithelial cells) and its mechanisms. Methods: ①Experimental protocol: control: the cells were treated with with 0 mg/L uric acid for 24 h; model: the cells were treated with with 1 200 mg/L uric acid for 24 h; pretreatment with iptakalim: the cells were pretreated with 0.01,0.1,1,10,100 μmol/L iptakalim for 24 h prior to treatment with 1 200 mg/L uric acid for 24 h; pretreatment with glibenclamide: the cells were preincubated with/without 10 μmol/L glibenclamide for 1 h and then treated with 10 μmol/L iptakalim for 24 h followed by incubation with 1 200 mg/L uric acid for another 24 h. ②The cell viability was measured by MTT assay and flow cytometry; the protein expressions of Kir6.1 and SUR2B and nuclear translocation were detected by immunostaining; the protein expressions of Kir6.1 and SUR2B were determined by Western blot analysis; adhesion of mononuclear cells to endothelial cells were tested by fluorimetric assay; the content of MCP-1 was measured by enzyme linked-immunosorbent assay (ELISA). Results: The renal glomerular endothelial, mesangial and tubular epithelial cells were exposed to 1 200 mg/L uric acid for 24 h. Compared with the control group, 1 200 mg/L uric acid decreased the cell survival rates significantly (P＜0.01, P＜0.01, P＜0.01). Compared with the model group, pretreatment with 0.1, 1, 10, 100 μmol/L iptakalim could remarkably alleviate cellular damages of glomerular endothelium, mesangium cells induced by uric acid (P＜0.05, P＜0.01, P＜0.01, P＜0.01). The K_ATP channel blocker could clearly reduce survival rates of the renal glomerular endothelial, mesangial cells(P＜0.01) and markedly reverse the inhibitory effects of iptakalim on cell death (P＜0.05, P＜0.01), no obvious difference in comparison with the model group (P＞0.05). Compared with the model group, pretreatment with 10, 100 μmol/L iptakalim could notably attenuate cellular damages of tubular epithelial cells induced by uric acid (P＜0.05, P＜0.05). The K_ATP channel blocker could obviously damage the tubular epithelial cells (P＜0.01), no obvious difference in comparison with the model group (P＞0.05). Compared with control group, exposure of renal tubular epithelial, mesangial and glomerular endothelial cells to 1 200 mg/L uric acid for 24 h caused a significant increase in the protein expressions of Kir6.1 and SUR2B(P＜0.05). Compared with the model group, the overexpressions of Kir6.1 and SUR2B were suppressed in presence of iptakalim at a concentration of 10 μmol/L (P＜0.05). These decreases in the expressions of Kir6.1 and SUR2B were prevented by the K_ATP channel blocker, no obvious difference in comparison with the model group (P＞0.05). Compare with the control group, monocytic adhesion to renal glomerular endothelial cells was notably promoted by 1 200 mg/L uric acid for 24 h (P＜0.01). Pretreatment with 10 μmol/L iptakalim for 24 h significantly reduced the monocytic adhesion in comparison with the model group (P＜0.05). It was showed that the inhibitory effects of iptakalim were antagonized by the K_ATP channel blocker, no obvious difference in comparison with the model group (P＞0.05). After stimulating glomerular endothelial cells with 1 200 mg/L uric acid for 24 hours, the secretion of MCP-1 was significantly increased compared to the control group (P＜0.05). Compare with the model group, preincubation with 10 μmol/L iptakalim significantly decreased MCP-1 production (P＜0.05). K_ATP channel blocker suppressed the downregulation of MCP-1 protein synthesis induced by iptakalim. After stimulation with uric acid, translocation of NF-κB from cytoplasms to nuclei of renal glomerular endothelial cells were observed, while that of NF-κB was suppressed in presence of iptakalim at the concentration of 10 μmol/L. This inhibition of NF-κB translocation was clearly prevented by K_ATP channel blocker. Conclusion: These results suggests that a new SUR2B/Kir6.1-type K_ATP channel opener iptakalim plays interventional roles in renal cells damages caused by uirc acid and its mechanism is involved in activating K_ATPchannels .

Key words: SUR2B/Kir6.1-type, ATP-sensitive potassium, iptakalim, renal injury, hyperuricemia

中图分类号:

R322.61

赵颖, 汪海. 激活SUR2B/Kir6.1亚型K_ATP通道对肾脏细胞损伤的干预作用及其机制^*[J]. 中国应用生理学杂志, 2022, 38(6): 604-610.

ZHAO Ying, WANG Hai. Interventional effects of activating SUR2B/Kir6.1-type K_ATP channels on renal cells injury and its mechanisms[J]. CJAP, 2022, 38(6): 604-610.

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