GSK3β调节Nrf2和胰岛素信号转导通路参与糖尿病肾病进展的机制研究

Microalbuminuria is the earliest clinical manifestation of diabetic nephropathy, in which podocyte dysfunction or injury has been centrally implicated. Evidence suggests that oxidative stress as well as defective insulin signaling, due to insulin deficiency or resistance, is likely a crucial initiator for podocyte injury in DN. Upon oxidative injury, nuclear factor erythroid 2-related factor 2 (Nrf2), the master regulator of antioxidant response and self-defense, is activated and harnessed for self-protection. Burgeoning evidence suggests that glycogen synthase kinase (GSK) 3β is centrally implicated in switching off the Nrf2 antioxidant stress response following injury by dictating Nrf2 nuclear exclusion and degradation.In addition, GSK3β has been originally characterized to be the key transducer of insulin signaling pathway. Our preliminary data demonstrated that the GSK3β regulated Nrf2 signaling is involved in podocyte response to Adriamycin-elicited injury. Here, we propose to continue our study and examine the role of GSK3β mediated Nrf2 and insulin signaling pathways in podocyte injury and proteinuria in DN. In kidney biopsy tissues from DN patients, the expression and activities of GSK3β and the related Nrf2 and insulin signaling pathways will be examined in glomeruli and podocytes. Results will be correlated with clinical and histologic parameters, including albuminuria, kidney dysfunction and glomerular injury. In cultured podocytes exposed to high glucose, the activity of GSK3β will be artificially manipulated by RNA interference or by transfection with plasmids encoding mutant GSK3β, and changes in Nrf2 response and insulin signaling will be assessed and correlated with podocyte injury. To examine the role of podocyte specific GSK3β in podocyte injury in DN in vivo, genetic targeting of GSK3β by doxycycline-inducible podocyte-specific knockout or pharmacologic targeting by SB216763, a highly selective small molecule inhibitor of GSK3β will be implemented in db/db or streptozotocin-induced diabetic mouse models. The effect of targeting GSK3β on Nrf2 response as well as insulin signaling in podocytes and on signs of DN will be determined. Collectively, our study will provide a mechanistic view of the role of podocyte specific GSK3β in podocyte oxidative injury and insulin signaling deficiency in diabetic nephropathy and may pave the way for testing a novel therapeutic target for protecting podocytes and treating DN.

胰岛素信号通路紊乱和氧化应激损伤是糖尿病肾病（DN）的重要致病机制。1)胰岛素信号通路对维持肾稳态有重要作用，糖原合成酶激酶3β（GSK3β）是该通路的关键信号转导分子。前期研究发现DN足细胞中GSK3β表达增加且活性增强,高糖促进小鼠足细胞GSK3β表达增加,且人尿沉渣脱落细胞GSK3β表达与DN进展相关,这些都提示GSK3β是DN进展的关键分子。2)GSK3β参与调节转录因子NF-E2相关因子2（Nrf2）所介导的抗氧化反应，前期研究发现GSK3β调控的不依赖于Keap1的Nrf2通路可有效缓解阿霉素所致足细胞损伤。而DN中, GSK3β是否通过调控Nrf2通路和胰岛素通路介导足细胞损伤尚不明确,以GSK3β为靶点的药物是否能够缓解DN也亟需证明。本课题从人体、细胞和动物三个方面,采用基因和药物靶向干预,系统探索GSK3β通过调节Nrf2及胰岛素通路参与DN足细胞损伤的作用机制。

足细胞损伤是导致糖尿病肾病发生发展的重要原因。GSK3β在糖尿病肾病足细胞损伤中表达及活性发生增强，而其是否参与足细胞损伤及相关作用机制还尚不明确。研究显示足细胞氧化应激损伤与胰岛素抵抗是足细胞损伤的重要指标，而GSK3β是调节Nrf2和胰岛素抵抗的关键分子。因此本项目通过收集临床标本及数据、体外培养足细胞以及利用糖尿病肾病小鼠模型，系统探讨了GSK3β-Nrf2/胰岛素信号通路在糖尿病肾病中发展的作用。为进一步延续该项目以及为临床转化作铺垫，我们也探索了中药单体对糖尿病肾病的作用以及是否通过调节Nrf2/胰岛素信号通路发挥作用。主要研究结果如下：（1）糖尿病肾病患者肾脏中GSK3β表达增加、胰岛素信号通路受损以及Nrf2核聚集增多，且与肾脏损伤程度相关；（2）GSK3β通过对Nrf2和胰岛素通路的调节参与高糖环境下足细胞的损伤；（3）中药单体大黄酸/迷迭香酸通过调节GSK3β-Nrf2/胰岛素通路参与调节糖尿病肾病的发展及机制研究。