基于内源性KIM-1/TIM-4通路探讨黄芪甲苷防治肾纤维化的机制

Persistent illness entering collaterals is the core pathogenesis of the renal fibrosis after acute kidney injury(AKI).Kidney injury molecular-1(KIM-1) is an early marker of AKI and its natural ligand TIM - 4 is highly expressed in macrophages.So far ,about the association between KIM - 1 and renal fibrosis,we still know very little.In our early studies , we found in the animal model of renal fibrosis that KIM-1 was expressed in local damaged renal tubule with macrophage infiltration and astragaloside can inhibit the expression of KIM - 1 and improve renal fibrosis. But the specific mechanism of strengthening the body resistance to eliminate pathogenic factors of astragaloside remains elusive. Therefore, we propose a hypothesis: the endogenous KIM - 1 may promote renal fibrosis through the KIM - 1 / TIM - 4 siginal, namely vital Qi deficiency and pathogen usually intruding into collaterals in protracted disease ,while the tonifying kidney-qi of astragaloside can regulate the siginaling pathway. To test this hypothesis, we adopt the immunohistochemical, confocal fluorescence microscope technology, real time PCR, Western blot, gene knockout and other mehtods to explore the mechanism of local kidney immune inflammation reaction mediated by KIM - 1 / TIM - 4 siginal in vivo and in vitro, respectively. In this study,we will further clarify the renal fibrosis siginaling of KIM - 1 binding to the ligand TIM - 4 and inducing macrophages secreteing inflammatory factors. From the new perspective of KIM - 1 and immune inflammation, this study will provide important insights into revealing the pathogenesis of renal fibrosis and theoretical foundation for further exploring the mechanism of tonify kidney-qi and restoring blood vessels and collaterals flow of astragaloside to prevent renal fibrosis.

久病入络是急性肾损伤后肾纤维化核心病机。KIM-1是肾损伤早期标记物，其天然配体TIM-4高度表达于巨噬细胞。迄今对KIM-1与肾脏纤维化关系仍知甚少。前期研究在肾纤维化动物模型受损肾小管中发现，KIM-1表达局部伴巨噬细胞浸润；以黄芪甲苷的益气通络作用能够抑制KIM-1表达，可改善肾纤维化。但黄芪甲苷调节免疫的具体机制仍待探讨。故推测：内源性KIM-1可能通过KIM-1/TIM-4通路促进肾纤维化，即正气亏虚，久病入络，而黄芪甲苷可对该通路起调控作用，延缓或阻断肾纤维化，达到益气通络而扶正。 本研究采用分子生物学方法和基因敲除手段，从体内、外实验探索KIM-1/TIM-4通路介导肾脏局部免疫炎症反应的机制。明确KIM-1结合TIM-4诱导巨噬细胞分泌炎症因子进一步调控导致肾纤维化的途径。实验将以KIM-1与免疫炎症为切入点，揭示肾纤维化发生、发展机制，探讨黄芪甲苷防治肾纤化的作用靶点。

KIM-1 是肾损伤早期标记物，前期研究发现，在肾纤维化动物模型受损肾小管中KIM-1 表达局部伴巨噬细胞浸润；以黄芪甲苷(AS-IV)的益气通络作用能够抑制 KIM-1 表达，可改善肾纤维化。本研究通过体内和体外实验探讨AS-IV防治肾纤维化的具体机制。研究结果明确KIM-1/TIM-4 通过丝裂原活化蛋白激酶(MAPK)信号通路调节巨噬功能来介导肾损伤的发生发展。KIM-1可通过抑制MAPK信号通路活化，促进巨噬细胞TIM-4表达水平增加，促进巨噬细胞活化和迁移，参与肾损伤纤维化的过程。AS-IV作为中药黄芪的主要有效成分之一，通过MAPK信号通路抑制KIM-1表达，减少巨噬细胞迁移和活化，调节肾脏巨噬细胞亚型，改善肾纤维化水平。本研究发现KIM-1/TIM-4信号通路在肾损伤中的重要作用，以及巨噬细胞和MAPK信号通路在其中具体的作用机制，全面阐述了AS-IV通过KIM-1/TIM-4通路防治肾纤维化的具体机制，为AS-IV的临床应用提供实验依据。