Smad3磷酸化抑制剂（SIS3）延缓ACE2基因敲除小鼠慢性肾衰竭的保护作用及机制研究

Chronic kidney disease (CKD) is becoming a world problem, but mechanisms remain unclear. Thus，it is the urgent need to define the molecular mechanisms and develop effective therapy for CKD. Recently, we found that levels of intra-renal Ang II are significantly increased after unilateral ureteral obstruction (UUO) in ACE2 knockout mice. This is associated with that the over-activation of local renin-angiotensin system (RAS) due to the impairment of the angiotensin II (Ang II) degradation pathway. We also found that Ang II-induced renal fibrosis and inflammation are mediated through the TGF-β/Smad3 pathway because mice lacking Smad3 are protected against Ang II-induced progressive kidney injury. These findings lead us to hypothesize that Smad3 may be a key mediator in the development of CKD associated with activation of local RAS and that specific inhibition of Smad3 may be a novel and effective therapy for CKD. The hypothesis will be examined in this study. We will first determine the mechanisms that activation of intrarenal RAS promotes the development of chronic renal failure in 5/6 nephrectomy mice induced in ACE2 gene knockout mice. Then, we will investigate the protective role and therapeutic potential in 5/6 nephrectomy mouse model induced in ACE2 gene knockout mice by blocking Smad3 with a specific Smad3 inhibitor (SIS3). Outcomes from this study will identify new mechanisms that loss of ACE2 promotes CKD by enhancing local activation of RAS and activation of both TGF-β/Smad3 and NF-κB signaling pathways. The therapeutic efficacy of SIS3 on the 5/6 nephrectomy mice will provide new evidence for novel treatment for CKD by targeting Smad3 signaling.

慢性肾脏病（CKD）已成为一个世界性问题，但机制仍不清楚，亟需研究CKD进展的机制和治疗策略。最近，我们发现ACE2基因敲除小鼠单侧输尿管结扎后肾脏血管紧张素-II（Ang II）显著增高，与破坏Ang II降解通路引起局部RAS系统过度激活有关，而Ang II诱导的肾损害是TGF-β/Smad3通路介导的，Smad3基因敲除小鼠不发生Ang II介导的肾损害，提示Smad3的激活可能是肾内RAS活化促进CKD进展的一个关键机制。因此提出假说：抑制Smad3磷酸化在慢性肾衰竭中具有治疗的潜能，并分两步验证假说：①在ACE2基因敲除小鼠中建立5/6肾切除慢性肾衰竭模型，研究ACE2在局部RAS激活和RAS-TGF-β/Smad3轴对CKD进展的作用及可能机制。②在ACE2基因敲除小鼠5/6肾切除模型，应用SIS3探索治疗CKD的新方法。本研究将为以Smad3为靶点治疗CKD提供新的证据。

慢性肾脏病患病率高，知晓率低，是一个世界性的公共卫生问题，但慢性肾脏病进展的机制仍不清楚,亟需研究CKD进展的机制和治疗策略。肾素血管紧张素系统（RAS）的过度激活在慢性肾脏病（CKD）的进展中发挥重要作用，血管紧张素II（AngII）是RAS的主要效应因子，AngII产生和降解的平衡在慢性肾脏病中意义重大..最近，我们发现ACE2基因敲除小鼠单侧输尿管结扎后肾脏血管紧张素-II（AngII）显著增高，与破坏AngII降解通路引起局部RAS系统过度激活有关，而AngII诱导的肾损害是TGF-β/Smad3通路介导的，Smad3基因敲除小鼠不发生AngII介导的肾损害，提示Smad3的激活可能是肾内RAS活化促进CKD进展的一个关键机制。因此提出假说：抑制Smad3磷酸化在慢性肾衰竭中具有治疗的潜能，并在ACE2基因敲除小鼠中建立5/6肾切除慢性肾衰竭模型，探讨ACE2在局部RAS激活和RAS-TGF-β/Smad3轴对CKD进展的作用及可能机制。实验发现，与野生型小鼠模型组相比，ACE2基因敲除小鼠血压增高（p＜0.05），肾损伤加重，表现为蛋白尿和血肌酐的升高（p＜0.05），肾脏纤维化和炎症指标加重，TGF-β/Smad3和NF-κB信号通路激活。另外，升高的Smurf2介导的Smad7蛋白的泛素化降解加重了敲除ACE2基因后5/6肾切除小鼠的肾脏纤维化和炎症。.因为Smad3的磷酸化对TGF-β/Smad和NF-κB通路发挥正调控作用，我们继而在ACE2基因敲除小鼠5/6肾切除模型，应用SIS3抑制剂验证了抑制Smad3的磷酸化能够阻止5/6肾切除术后肾损伤的进展，本研究将为以Smad3为靶点治疗CKD提供新的证据和思路。