背景:脓毒症相关急性肾损伤(SA-AKI)死亡率高。骨保护素(OPG)/核因子-κB受体激活因子配体(RANKL)/核因子-κB受体激活因子(RANK)/Toll样受体4(TLR4)通路及其在SA-AKI发病机制中的潜在作用仍有待充分了解。在这里,我们用鼠标模型解决了这个问题。
方法:使用盲肠结扎穿孔法(CLP)建立SA-AKI小鼠模型。小鼠被分组为假,CLP模型,CLP+重组RANKL,和CLP+抗RANKL组。测量血清肌酐(Scr)和血尿素氮(BUN)水平以评估肾功能。ELISA法检测血清IL-1β,TNF-α,和IL-6水平。采用实时定量PCR和Westernblot检测OPGmRNA和蛋白表达水平,RANKL,RANK,和肾组织中的TLR4。HE染色评价病理改变。
结果:CLP模型组显示出更高水平的Scr和BUN,表明SA-AKI肾功能受损,与假手术组相比。CLP+重组RANKL组用重组RANKL处理可降低Scr和BUN水平,而CLP+抗RANKL组的抗RANKL治疗提高了它们的水平。此外,CLP模型组IL-1β明显升高,TNF-α,和IL-6比假手术组,表明SA-AKI的炎症升高。CLP+重组RANKL组显示细胞因子水平降低,而CLP+抗RANKL组显示增加。此外,组织病理学评估显示CLP模型组明显的肾组织损伤.重组RANKL治疗减少了这种损伤,而抗RANKL治疗加剧了这种情况。机械上,RANKL的mRNA和蛋白表达显著降低,而那些OPG,RANK,CLP模型组和CLP+抗RANKL组TLR4明显升高。有趣的是,用重组RANKL治疗逆转了这些变化,如RANKL显著增加但OPG降低所证明,RANK,TLR4
结论:OPG/RANKL/RANK/TLR4通路参与SA-AKI发病机制。重组RANKL治疗减轻SA-AKI的炎症反应和肾组织损伤,可能是通过调节这个途径。该途径显示出有望作为SA-AKI的治疗靶标。
BACKGROUND: Sepsis-associated acute kidney injury (SA-AKI) has high mortality rates. The osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of nuclear factor-κB (RANK)/Toll-like receptor 4 (TLR4) pathway and its potential role in SA-AKI pathogenesis remain to be fully understood. Herein, we addressed this issue using mouse models.
METHODS: An SA-AKI mouse model was established using the cecal ligation and puncture method (CLP). Mice were grouped into sham, CLP model, CLP + recombinant RANKL, and CLP + anti-RANKL groups. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured to assess kidney function. ELISA was used to detect serum IL-1β, TNF-α, and IL-6 levels. Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression levels of OPG, RANKL, RANK, and TLR4 in kidney tissues. HE staining was performed to evaluate the pathological changes.
RESULTS: The CLP model group showed higher levels of Scr and BUN, indicating impaired kidney function in SA-AKI, compared to the sham group. Treatment with recombinant RANKL in the CLP + recombinant RANKL group reduced Scr and BUN levels, while anti-RANKL treatment in the CLP + anti-RANKL group elevated their levels. Moreover, the CLP model group had significantly increased IL-1β, TNF-α, and IL-6 than the sham group, indicating elevated inflammation in SA-AKI. The CLP + recombinant RANKL group demonstrated decreased cytokine levels, whereas the CLP + anti-RANKL group showed an increase. Additionally, the histopathological evaluation revealed distinct kidney tissue damage in the CLP model group. Recombinant RANKL treatment reduced this damage, while anti-RANKL treatment exacerbated it. Mechanically, the mRNA and protein expression of RANKL were significantly decreased, while those of OPG, RANK, and TLR4 were significantly increased in the CLP model group and the CLP + anti-RANKL group. Interestingly, treatment with recombinant RANKL reversed these changes, as evidenced by significantly increased RANKL but decreased OPG, RANK, and TLR4.
CONCLUSIONS: The OPG/RANKL/RANK/TLR4 pathway is involved in SA-AKI pathogenesis. Recombinant RANKL treatment attenuates the inflammatory response and kidney tissue damage in SA-AKI, possibly via regulating this pathway. This pathway shows promise as a therapeutic target for SA-AKI.