MFG-E8在糖尿病肾病蛋白尿和足细胞凋亡中的作用及分子机制

Diabetic nephropathy (DN), as one of the most feared chronic microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. Moreover, clinicians face an approximately 30% prevalence of patients with type 2 DM (T2DM) and DN. An early sign of DN is an increased amount of albuminuria, which correlates with, and can predict, the progression of renal damage. Albuminuria arises from defects in the permeability of the glomerular filtration barrier, which is mainly caused by the abnormal apoptosis of podocytes under the high glucose、AGEs and TGF-β. .In Our previous studies，proteome analysis by isobaric tags for relative and absolute quantification (iTRAQ) identified MFG-E8 is highly expressed in diabetic heart, kidney, aorta, and retina. Over-expression of MFG-E8 in diabetic kidney significantly exacerbated albuminuria, renal histological and ultrastructural lesions in db/db mice. In the contrast, MFG-E8 RNA silence significantly improved renal histological lesions. But, the underlying molecular mechanism is unclear. Cytoskeleton is important for podocyte structure and function . Actin cytoskeleton reorganization and redistribution relates to podocyte apoptosis and albuminaria. It is reported that MFG-E8 derived short peptide 68 (MSP68) inhibits neutrophil migration by modulating small GTPase Rac1e dependent cytoskeletal rearrangements. Hence, we supposed that MFG-E8 may aggravate aluminuria by regulating actin cytoskeleton and podocyte apoptosis..Here, we transfect LV-MFG-E8 and LV-MFG-E8 RNAi targeting to db/db mice kidney, to up-regulate or down-regulate RNA and protein expression of MFG-E8 in diabetic kidney. We test fasting blood glucose (FBG), AGEs, renal function including blood urea nitrogen (BUN), plasma creatinine concentration (Cr) and urinary albumin excretion rate in mice, to evaluate the effect of MFG-E8 on glomerular filtration barrier. TUNEL in situ kit and Hoechst 33342 are applied to evaluate the effects of MFG-E8 on podocyte apoptosis. Confocal immunofluorescence, immunoprecipitation , RT-PCR, western blot are also used in this study. We hope this study could provide a new insight into the pathogenesis of diabetic nephropathy and targeting MFG-E8 could be a novel strategy to retard the progression of DN.

DN是造成终末期肾脏病的主要原因。DN的早期表现是蛋白尿的产生。足细胞异常凋亡是造成蛋白尿的病理基础。我们的前期研究表明，MFG-E8的过表达加重糖尿病肾病损害。但具体机制不明。MFG-E8通过影响肌动蛋白相关蛋白2/3的再定位、肌动蛋白张力纤维的溶解以及新的片状伪足的建立，调控细胞的迁移。MFG-E8调控Rac1-MAPK丝状肌动蛋白依赖的细胞骨架的变形。因此，我们推测“MFG-E8可能通过调控肌动蛋白细胞骨架的稳定性导致足细胞的异常凋亡，加重蛋白尿的产生”。我们拟采用db/db小鼠为糖尿病肾病模型，采用慢病毒载体靶向干预肾脏MFG-E8基因表达， ELISA检测24小时尿白蛋白排泄率、TUNEL原位检测足细胞凋亡率，以及Hoechst 33342染色、免疫荧光、免疫共沉淀、RT-PCR等技术，从动物水平和细胞水平两个层面进行验证。此研究将为糖尿病肾病的诊疗提供新的诊疗策略。

DN是造成终末期肾脏病的主要原因，早期表现为蛋白尿的产生。足细胞异常凋亡是造成蛋白尿的病理基础。MFG-E8是亲脂性糖蛋白，可以与细胞膜上的整合素受体结合，参与调控细胞骨架的稳定性及细胞增殖、衰老及凋亡等生物过程。然而，MFG-E8在糖尿病足细胞损伤中的作用及分子机制尚不明确。我们前期蛋白质组学结果表明，在糖尿病肾组织中，MFG-E8呈高表达，而减低MFG-E8基因表达后其肾组织损伤减轻。因此，我们推测MFG-E8可能通过调控胞骨架的稳定性影响足细胞的异常凋亡，加重蛋白尿的产生。本项目在前期研究基础上，从体内实验及体外实验两个方面，通过慢病毒载体靶向干预MFG-E8基因表达， 采用TUNEL、免疫荧光、免疫共沉淀、RT-PCR等技术，明确了MFG-E8可通过调控整合素-FAK信号通路影响f-actin细胞骨架的稳定性，参与调控足细胞线粒体氧化应激及足细胞异常凋亡，从而加重糖尿病状态下蛋白尿的生成。另外，我们在实验过程中发现高糖诱导下的脂质聚集是造成足细胞损伤的关键原因之一，左卡尼汀作为糖脂代谢的调节因子之一，可显著改善足细胞损伤，减少蛋白尿的生成。并从分子水平明确了左卡尼汀通过调控AMPK/PGC1a/CPT-1信号通路参与调控细胞骨架的稳定性及足细胞损伤。因此本研究揭示了脂质代谢紊乱和细胞骨架结构紊乱在足细胞损伤中的关键作用，为防治糖尿病肾病提供了研究思路。