Numb在肾脏细胞自噬中的作用及其机制研究

Acute kidney injury (AKI) is a major renal disease associated with significant mortality and the development of chronic kidney disease and end-stage renal disease. However, current treatment options are limited. Pathologically, AKI is characterized by tubular cell injury and death. Recent studies have demonstrated that autophagy is rapidly induced during AKI to protect tubular cells from injury and death. .Numb plays a critical role in cell endocytosis, adhesion, migration, differentiation, and self-renewal. Our previous studies showed that Numb was well expressed in renal tubular epithelial cells. In renal ischemia-reperfusion injury and cisplatin induced AKI mice, renal Numb protein expression was significantly upregulated. Our further studies showed that reduction of Numb could down-regulate p62 and up-regulate LC3II protein expression. These data suggested that reduction of Numb might promote renal autophagy. .Studies had shown cytosolic p53 inhibited autophagy, and Numb plays a role in p53 stabilization. Over-expression of Numb increases the level of p53, while reduction of Numb causes a decrease p53 expression. Our studies also showed that reduction of Numb could down-regulate p53 protein expression，suggesting that Numb might inhibite autophagy via p53 stabilization..We are going to validate this hypotheses by the following studies. Firstly, we are going to detect the changes of autophage in Numb over-expression and knock-down renal epithelial cells. Secondly, we are going to examine the changes of autophage in Numb over-expression cells when inhibiting p53 expression or Numb knock-down cells when inhibiting HDM2 expression. At last, we are going to confirm these changes in renal Numb gene knock-out mice.

急性肾损伤(AKI)增加住院患者死亡率和致残率，也是致慢性肾衰竭的重要原因，肾小管上皮细胞损伤是其主要病理表现。自噬在AKI中具有保护作用。Numb是一个高度保守的蛋白，具有多种生物学功能，但其在肾脏功能不清。我们发现，Numb在肾小管上皮细胞广泛表达，且在AKI时表达显著上升；沉默Numb导致p62降低，LC3II上升，提示下调Numb促进自噬。研究表明，胞浆p53抑制自噬，而Numb可与p53及HDM2结合形成三聚体，抑制p53降解。我们也发现，沉默Numb后p53显著降低；因此，我们提出假设：Numb通过抑制p53降解，增加p53蛋白水平，抑制细胞自噬，促进AKI损伤。我们将分别上调、下调Numb明确Numb对肾小管上皮细胞自噬的作用及其机制；建立肾脏Numb基因敲除小鼠，验证Numb通过抑制自噬加重AKI损伤的机制。本研究将进一步揭示AKI的损伤机制，为临床防治AKI提供新靶点。

研究背景：急性肾损伤(AKI)增加住院患者死亡率和致残率，也是致慢性肾衰竭的重要原因。Numb是一个高度保守的蛋白，具有多种生物学功能。我们前期研究发现，Numb在肾小管上皮细胞广泛表达，在AKI时表达显著上调，表明Numb在AKI中具有重要的作用。本项目旨在研究Numb在AKI中的作用及其机制。.主要研究内容和重要结果、关键数据：1、在肾小管上皮细胞上调或下调Numb表达，观察Numb在顺铂诱导细胞损伤中的作用，结果发现：Numb在顺铂刺激细胞后表达显著升高，Numb缺失后肾小管损伤更为严重，细胞凋亡水平更高；在肾小管上皮细胞Numb基因特异敲除小鼠（PT-Nb-KO）采用顺铂刺激和缺血再灌注建立AKI模型，观察Numb在AKI中的作用，结果发现：Numb在AKI高表达，Numb缺失后肾功能损伤更为严重，肾小管损伤加重，细胞凋亡加重；以上结果提示，Numb在AKI高表达，Numb缺失加重AKI损伤；2、采用顺铂刺激干扰Numb后的肾小管上皮细胞，采用顺铂刺激及缺血再灌注建立PT-Nb-KO小鼠AKI模型，检测线粒体损伤情况，结果发现：Numb缺失后，线粒体分裂的细胞数增多，线粒体的片段化加重，线粒体相关蛋白Drp1、Fis1表达显著升高，而OAP1、Mfn1/2表达显著降低，Drp1 Ser637位点磷酸化水平显著上调，体内体外实验结果相似；进一步应用线粒体损伤相关蛋白抑制剂预干预细胞或小鼠，观察线粒体损伤、片段化情况，结果发现应用Drp1抑制剂mdivi1预干预后，细胞线粒体的分裂减轻，凋亡减少；小鼠肾脏损伤显著减轻，细胞凋亡显著减少；以上结果提示：Numb缺失上调Drp1 Ser637磷酸化，增加线粒体Drp1募集，加重细胞线粒体损伤，加重AKI损伤。.科学意义：我们发现，与慢性肾脏病（CKD）相反，Numb在AKI中起着一定的保护作用，其作用可能通过调节Drp1蛋白减轻线粒体损伤来实现。本项目揭示了Numb在AKI过程中线粒体损伤的新机制，为AKI的防治提提供了新靶点。