去泛素化酶USP9X通过Connexin43抑制糖尿病肾小管间质纤维化的研究

Renal tubular epithelial-mesenchymal transdifferentiation (EMT) induced by hyperglycemia and transforming growth factor-β (TGF-β) is the critical pathogenesis of diabetic renal tubulointerstitial fibrosis, and Connexin43 (Cx43) is closely associated with EMT. Recent studies have demonstrated that ubiquitin-specific protease 9X (USP9X) can regulate the TGF-β signaling pathway. Whether USP9X influences the pathological development of renal EMT and tubulointerstitial fibrosis under diabetic conditions and the relationship between USP9X and Cx43 deserve further investigation. Our previous study have shown that USP9X is crucially related with diabetic renal tubulointerstitial fibrosis. USP9X upregulation inhibited the EMT process of NRK-52E cells exposed to high glucose (HG) and decreased the expression of extracellular matrix (ECM) including fibronectin (FN), which was correlated to deubiquitinating Cx43. Based on these foundations, the current project was undertaken to confirm the regulatory effect of USP9X on diabetic renal tubulointerstitial fibrosis with the exploitation of EMT markers and ECM components in both in vivo diabetic animal kidneys and in vitro NRK-52E/HK-2 cells challenged with HG. Then, from the perspective of affecting Cx43 degradation by ubiquitin-proteasome pathway, further efforts have been made to explore the molecular mechanisms of the inhibitory effect of USP9X on diabetic renal tubulointerstitial fibrosis. Taken together, this work will provide objective experimental evidence for developing USP9X as a possible therapeutic target to delay the progression of diabetic nephropathy.

高血糖、TGF-β诱导的肾小管上皮细胞-间充质细胞转分化（EMT）是糖尿病肾小管间质纤维化的关键病理因素，缝隙连接蛋白Cx43与EMT关系密切。近年来有充分证据表明去泛素化酶USP9X具有调控TGF-β通路的作用。USP9X能否影响糖尿病状态下肾脏EMT及肾小管间质纤维化的病理进程，以及该作用与Cx43的关系，值得高度关注。我们前期研究显示：USP9X与糖尿病肾小管间质纤维化具有重要关联性；上调USP9X能够抑制高糖诱导的NRK-52E细胞发生EMT，并减少FN等ECM表达，且与去泛素化Cx43有一定关系。在此基础上，本项目拟通过研究USP9X对模型动物肾组织和模型细胞EMT标志分子及ECM表达的影响，明确USP9X对糖尿病肾小管间质纤维化的作用；从Cx43泛素-蛋白酶体通路降解角度，探讨USP9X阻抑糖尿病肾小管间质纤维化的分子机制，为将USP9X作为抗糖尿病肾病的潜在靶点提供实验依据。

高血糖等因素诱导的肾小管上皮细胞-间充质细胞转分化（EMT）是糖尿病肾小管间质纤维化的关键病理因素。本项目利用体内db/db糖尿病模型小鼠和体外高糖培养的肾小管上皮细胞NRK-52E细胞，首先证实缝隙连接蛋白（Cx43）通过其羧基末端增加Sirt1的表达，促进Sirt1对HIF-1α去乙酰化，降低HIF-1α蛋白表达以及转录活性，最终抑制EMT的发生以及糖尿病肾小管间质纤维化，揭示了Cx43抗糖尿病肾病新的作用机制。同时，本项目通过过表达和沉默泛素特异性蛋白酶9X（USP9X）的方法，观察高糖诱导的NRK-52E细胞中EMT标志分子以及FN等ECM成分表达的变化，明确了USP9X对糖尿病肾小管间质纤维化的抑制作用；从Cx43的泛素化-蛋白酶体降解角度切入，发现USP9X可通过直接去泛素化Cx43而减少其降解，增强Cx43抑制肾小管上皮细胞EMT发生的功能，从而逆转糖尿病肾小管间质纤维化的病变进程。本项目通过探讨USP9X影响糖尿病肾小管间质纤维化的机制，为糖尿病慢性微血管并发症发病机制提供新的理论认识，为探索将USP9X作为抑制糖尿病肾脏纤维化、抗糖尿病肾病作用的潜在靶点提供客观的实验证据，具有重要的研究价值和科学意义。