钠钙离子交换蛋白在对比剂致急性肾损伤中的作用及机制

Renal hemodynamic disorder and direct tubular toxicity are main mechanisms of contrast-induced acute kidney injury (CI-AKI). But little is known about the molecular mechanisms underlying CI-AKI. Intracellular Ca2+ overload is considered to be a key factor in contrast-induced renal tubular cell injury and renal hemodynamic disorder. Na+/Ca2+ exchanger (NCX) is a bidirectional plasma membrane transporter expressed in renal tubular epithelial cell that catalyzes the exchange of Na+ for Ca2+. Under physiological condition, NCX can pump the Ca2+ outside the cell using Na+ concentration gradient across the cell membrane to keep the low intracellular Ca2+ ([Ca2+]i) level. In pathological condition, NCX can reversely extrude Na+ for Ca2+ influx. Our previous study has shown that the inhibitor of reverse mode of the NCX attenuated contrast-induced renal tubular epithelial cell apoptosis through inhibiting the increase of [Ca2+]i. We postulate that contrast media induce the Ca2+ influx via the reverse mode of NCX, followed by Ca2+ release from the sarcoplasmic reticulum stores and result in the intracellular Ca2+ overload, which is the major molecular basis underlying contrast-induced renal hemodynamic disorder and direct tubular toxicity. In the present study, the effects of inhibitor and activator of reverse mode of NCX and NCX1 gene knockout on the changes of renal tubular epithelial cell apoptosis and NCX expression, [Ca2+]i, reactive oxygen species (ROS) and endothelin-1 production induced by contrast media are observed. The effects of NCX1 gene knockout and the inhibitor of reverse mode of NCX on the contrast media-induced changes in NCX expression, intrarenal endothelin-1 production,renal hemodynamics, renal function and renal histopathology are investigated in rats with CI-AKI. In summary, the new molecular mechanisms underlying CI-AKI and the new therapy target point against CI-AKI are investigated in the program.

肾血流动力学紊乱及直接肾小管毒性是对比剂致急性肾损伤的主要机制，但其分子机制并不明确。细胞内钙超载是对比剂致肾小管细胞损伤及肾血流动力学紊乱的关键因素，钠钙离子交换蛋白（NCX）是存在于肾小管上皮细胞供细胞膜内外钠、钙交换的双向质膜载体蛋白，其反向转运是病理状态下钙内流的主要通道。我们前期研究显示NCX反向模式抑制剂可抑制细胞内钙超载减轻对比剂诱导的肾小管细胞损伤。我们推测对比剂诱导NCX反向转运介导钙内流，促发胞内网膜系统钙释放致细胞内钙超载是对比剂致肾血流动力学紊乱及直接肾小管毒性的主要分子基础。本项目观察NCX反向模式抑制剂及激动剂、NCX1基因敲除等对对比剂诱导的肾小管细胞凋亡及NCX表达、细胞内钙、活性氧、内皮素生成等的影响；探讨抑制NCX反向转运或NCX1基因敲除对对比剂致急性肾损伤鼠的影响。意义：提出对比剂致急性肾损伤新的分子机制；为对比剂致急性肾损伤的防治提供新的治疗靶点。

该项目在天津医科大学与武汉大学同时进行，现已按时完成。共发表论文4篇，其中SCI论文1篇，中华论文2篇。重要结果：（1）碘普罗胺（低渗对比剂）与NRK-52E细胞（肾小管上皮细胞）共培养可诱导肾小管上皮细胞ROS增多，肾小管上皮细胞GRP78 及CHOP表达增加以及肾小管上皮细胞凋亡；(2）对比剂诱导的ROS增多是其诱导内质网应激的上游事件：抗氧化剂NAC可以通过减轻对比剂诱导的ROS增多抑制对比剂诱导的GRP78以及CHOP过表达，减轻对比剂诱导的肾小管上皮细胞凋亡；（3）碘普罗胺与NRK-52E细胞共培养可诱导肾小管上皮细胞凋亡和自噬，随着对比剂浓度增加肾小管上皮细胞自噬体表达以及肾小管上皮细胞LC3Ⅱ/ LC3 I的表达先上升后下降；小剂量对比剂刺激下肾小管上皮细胞自噬和凋亡同时发生，但高峰早于凋亡；（4）雷帕霉素与碘普罗胺共处理肾小管上皮细胞后，LC3Ⅱ/ LC3 I、Beclin-1及GFP—LC3表达上调，细胞凋亡减少；3-甲基腺嘌呤有相反结果；（5）高胆固醇饮食（HCD）8周诱导了血清总胆固醇浓度，肾血管阻力指数以及肾内MDA，COX-2表达增加，肾实质TXB2，PGE2含量也明显增加，且TXB2改变较PGE2改变更明显。HCD 8周大鼠注射对比剂可出现对比剂诱导的急性肾损伤（CI-AKI），正常饮食大鼠注射等剂量对比剂未出现血肌酐明显变化。高胆固醇血症加重对比剂肾损害的机制与其诱导肾组织COX-2过表达以及肾内前列腺素系统紊乱导致的肾血管收缩有关。（6）肾小管上皮细胞NCX反向转运介导的肾组织氧化应激增加及ET-1过度生成参与CI-AKI的发病；抑制肾小管上皮细胞NCX反向转运可通过抑制对比剂诱导的ET-1增多、减轻对比剂诱导的肾血管收缩、氧化应激损伤及肾小管上皮细胞凋亡对CI-AKI起保护作用。科学意义：（1）进一步揭示了对比剂致肾小管细胞凋亡的亚细胞及分子机制：ROS介导的内质网应激参与对比剂诱导的肾小管上皮细胞凋亡；自噬可通过减轻氧化应激对对比剂诱导的肾小管上皮细胞凋亡起保护作用；（2）证实了肾小管上皮细胞NCX反向转运介导的肾组织氧化应激损伤以及ET-1过度生成参与CI-AKI的发病；（3）为CI-AKI的防治提供新的治疗靶点，提出了以发病机制为基础的新的CI-AKI防治策略：如抑制NCX反向转运；抑制氧化应激；抑制内质网应激，增加自噬等。