天然反义LncRNA-ARAP1-AS1和ARAP1-AS2协同调控ARAP1在糖尿病肾病发病机制和诊断中的作用

Our previous studies found the expression of serum lncRNA-ARAP1-AS1 were gradually downregulated, lncRNA-ARAP1-AS2 was gradually upregulated and their sense strand gene ARAP1((ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 1) mRNA was gradually upregulated along with the progression of diabetes and diabetic nephropathy. The diagnosis signature combined with the serum expression levels of lncRNA-ARAP1-AS1 and ARAP1-AS2 present a good diagnostic efficacy in diabetes mellitus and diabetic nephropathy. In present study, the expression levels of lncRNA-ARAP1-AS1，ARAP1-AS2 and ARAP1 in serum, urine and kidney tissue of diabetic nephropathy patients, high glucose cultured podocytes, mesangial cells, endothelial cells and renal tubular epithelial cells, and type 2 diabetes db/db mice kidneys will be detected. The coordinately regulatory mechanism of ARAP1-AS1 and ARAP1-AS2 on ARAP1 will be investigated. The objective is to elucidate the role of ARAP1-AS1 and ARAP1-AS2 on Rho/Cdc42, EGFR and its downstream pathway including PI3K/Akt/mTOR, ERK1/2, TGFβ1/Smad2/3, AMPK by upregulating ARAP1 to promote renal innate cells cytoskeleton rearrangement, autophagy inhibition, apoptosis, EMT, oxidative stress, endoplasmic reticulum stress, inflammation and fibrosis in diabetic nephropathy. The protective role of abated renal abnormal ARAP1-AS1, ARAP1-AS2 and ARAP1 expression on diabetic kidney injury will be observed. Furthermore, the value of diagnosis signature combined with the serum and urine lncRNA-ARAP1-AS1 and ARAP1-AS2 expression as biomarkers for diabetic kidney injury will be evaluated.

在前期实验基础上，验证糖尿病肾病（DN)患者血清、尿液和肾组织、体外培养的足细胞、系膜细胞、内皮细胞和肾小管上皮细胞、2型糖尿病db/db小鼠肾脏天然反义lncRNA-ARAP1-AS1、ARAP1-AS2及其正义链基因ARAP1的表达变化，探讨ARAP1-AS1和ARAP1-AS2对ARAP1的协同调控机制。明确ARAP1-AS1和ARAP1-AS2能否通过调控糖尿病状态下ARAP1的过表达，激活下游信号通路Rho/Cdc42和EGFR介导的PI3K/Akt/mTOR、ERK1/2、TGF-β1/Smad2/3、AMPK通路，促进肾脏固有细胞骨架重排、自噬抑制、凋亡、EMT、氧化应激、内质网应激、炎症和纤维化，探讨靶向抑制ARAP1-AS1和ARAP1-AS2异常表达能否减轻糖尿病肾脏损伤。验证结合血清、尿液ARAP1-AS1和ARAP1-AS2的诊断标签能否成为DN无创性生物标志物

本课题验证了糖尿病状态下肾脏固有细胞（系膜细胞和肾小管上皮细胞）、2型糖尿病db/db小鼠肾脏天然反义lncRNA-ARAP1-AS2及其正义链基因ARAP1的表达变化，探讨ARAP1-AS2对ARAP1的调控机制。首次获得人近端肾小管上皮细胞中天然反义lncRNA-ARAP1-AS2的全长序列，证明天然反义lncRNA-ARAP1-AS2通过与ARAP1的mRNA结合，顺式调控ARAP1的表达，同时可通过YY1反式调控ARAP1的作用机制。明确糖尿病状态下，天然反义lncRNA-ARAP1-AS2通过上调ARAP1的表达，增加了ARAP1与CIN85的结合，可能通过减少CIN85与泛素连接酶（E3）Cbl的结合，降低EGFR的泛素化水平，因此提供了更多可被高糖刺激的总EGFR的蛋白，从而维持了高糖状态下EGFR/TGF-β/Smad3信号传导通路的持续激活，促进糖尿病状态下近端肾小管上皮细胞损伤和细胞外基质蛋白积聚。同时也明确了糖尿病状态下ARAP1可上调Cdc42-GTP的水平，导致细胞骨架重排，细胞活力和迁移增强。靶向抑制天然反义lncRNA-ARAP1-AS2及其正义链靶基因ARAP1可以通过增加EGFR的泛素化抑制EGFR的持续性反式激活，减轻糖尿病状态下肾脏固有细胞损伤和细胞外基质蛋白积聚，并且通过抑制Cdc42-GTP的水平，减少肾小管上皮细胞细胞骨架重排、细胞活力、迁移过程、EMT以及纤维化标志蛋白的表达，为糖尿病病肾病的治疗提供新的思路。验证了钠-葡萄糖共转运体2(SGLT2)抑制剂恩格列净通过调控HIF-1α对高血糖诱导的肾小管上皮细胞损伤的保护作用，并且发现caspase-3抑制剂VX-765和Z-DEVD-FMK可显著改善糖尿病小鼠肾脏病理损伤，同时进一步从巨噬细胞和炎症的角度出发，为治疗糖尿病肾病提供新的方向和新的靶点。综上所示，本研究为糖尿病肾病的发病机制及靶向治疗提供了新的思路与方向。