外泌體在糖尿病腎病變中扮演的角色

Abstract

糖尿病是腎衰竭的主要原因之一，據統計國內目前的洗腎病人中約有40%-50%是糖尿病引起的。糖尿病腎病是糖尿病小血管病發症之一，它的進程涉及腎小球超濾、白蛋白尿、腎小球過濾速率下降，並最終導致腎衰竭。而糖尿病所引起的代謝變化將導致腎小球發炎、肥大和硬化以及腎小管間質發炎和纖維化。外泌體(Exosomes)是一種直徑界於50‐200奈米的囊泡，內含mRNA、miRNA、蛋白質及核酸等物質，並被證實會參與在糖尿病疾病進程之中，影響β-cell存活、葡萄糖不耐受性及和胰島素阻抗等。然而關於糖尿病血液中的外泌體是否在糖尿病腎臟病的進程中扮演重要的角色目前尚待釐清。因此本研究欲探討糖尿病血液中的外泌體是否會引起腎臟產生病變。本研究中 in vitro部分，我們將利用MTT assay及Western blots探討小鼠腎膈細胞(SV40-MES13)、小鼠腎小球內皮細胞(C57BL/6 mouse primary kidney glomerular endothelial cells)與大鼠近曲小管上皮細胞(NRK-52E)暴露於糖尿病db/db小鼠血液中的外泌體24小時後，觀察各別細胞之存活率及發炎、纖維化、EMT (epithelial-mesenchymal transition)、MFT (myofibroblast transdifferentiation)與EndMT (endothelial-mesenchymal transition) 相關蛋白表現情況。而在in vivo部分，我們將糖尿病db/db小鼠血液中的外泌體以尾靜脈注射方式送入對照組db/m小鼠體內，並將小鼠分成2天及14天後犧牲的組別，觀察糖尿病db/db小鼠血液中的外泌體對於正常db/m小鼠腎臟的影響。截至目前為止，在in vitro的部分，我們發現糖尿病db/db小鼠血液中的外泌體會: (1)促進小鼠腎膈細胞的增生，並顯著增加細胞中與MFT和纖維化相關蛋白的表現量; (2)顯著增加小鼠腎小球內皮細胞中與發炎和EndoMT相關蛋白的表現量; (3)顯著增加大鼠近曲小管上皮細胞中與EMT和纖維化相關蛋白的表現量。而在in vivo部分，我們也發現以尾靜脈將糖尿病db/db小鼠血液中的外泌體送入正常db/m小鼠後，在2天及14天後犧牲的兩者組別中: (1)小鼠血中尿素氣、肌酸酐和胱抑素C都顯著上升了; (2)小鼠腎臟組織中與發炎、纖維化、EMT、MFT與EndoMT相關蛋白的表現量都呈現增加的趨勢; (3)H E與Masson’s trichrome組織切片染色的結果發現小鼠腎小球出現肥大與細胞外基質沉積的現象，而在腎小管間質的部位也可觀察到細胞外基質沉積的現象; (4)螢光染色結果發現小鼠腎小球中CD31和Podocin的表現有下降的趨勢，並發現腎小球中細胞外基質Collagen IV的表現呈現增加的趨勢。接著我們透過次世代定序相關的生物分析資料預測並以qPCR驗證，推測外泌體可能是透過miR-669c-3p去抑制FOXO1基因的表現，進一步促進腎臟纖維化的產生。另外，以qPCR及western blotting進一步分析亦發現糖尿病db/db小鼠血液中的外泌體送入正常db/m小鼠之後，其腎臟中FOXO1的基因與蛋白表現量皆顯著下降。總結而言，在糖尿病db/db小鼠血液中的外泌體能引起腎病變，並發現外泌體中具有大量的miR-669c-3p，可調節腎臟中FOXO1的基因與蛋白表現，進一步促進腎臟纖維化。

Diabetes is the main causes of kidney failure, and there are about 40% to 50% of hemodialysis patients with diabetes in Taiwan. Diabetic nephropathy is a serious microvascular complication of diabetes. The progression of diabetic nephropathy involves in glomerular hyperfiltration, progressive albuminuria, declining GFR, and and ultimately leading to kidney failure. Metabolic changes associated with diabetes lead to glomerular hypertrophy, glomerulosclerosis, and tubulointerstitial inflammation and fibrosis. Exosomes are extracellular vesicles with 50‐200 nm in size. They contain mRNA, miRNA, proteins and nucleic acids, which have been proved to participate in the progression of diabetes. However, whether diabetic serum exosomes play a vital role in nephropathy remains unknown. Therefore, the objective of our study is to investigate whether diabetic serum exosomes induce kidney injure. In vitro study, we treated mouse mesangial cells (SV40-MES13), mouse primary kidney glomerular endothelial cells and rat proximal tubular epithelial cells (NRK-52E) with serum exosomes extracted from control db/m mice or diabetic db/db mice. In vivo study, we exposed db/m mice with serum exosomes isolated from db/m or db/db mice by tail vein injection. In vitro study, the administration of db/db mice serum exosomes compared to db/m serum exosomes induced: (1) mouse mesangial cells proliferation, MFT and fibrosis phenotype; (2) mouse primary kidney glomerular endothelial cells EndMT and inflammation phenotype; (3) rat proximal tubular epithelial cells EMT and fibrosis phenotype. In vivo study, we revealed that the db to m experimental mice (db/m mice tail vein injection with serum exosomes derived from db/db mice) compared to m to m control mice (db/m mice tail vein injection with serum exosomes derived from db/m mice) in 2 and 14 days sacrificed groups both improve: (1) their blood urea nitrogen (BUN), serum creatinine (SCr) and Cystatin C levels; (2) proteins expression related to inflammation, fibrosis, MFT, EndMT and EMT in kidney tissue; (3) glomerulus hypertrophy, glomerular and tubulointerstitial compartments displaying extracellular matrix collagen deposition detected by H E and Masson’s trichrome staining; (4) decreased level of CD31 and Podocin in glomerulus, and increased level of Collagen IV in glomerulus. Though Next Generation Sequencing (NGS) and qPCR analysis, we speculated that exosomes enriched in miR-669c-3p which can regulate the expression level of FOXO1 in kidney, and further promote renal fibrosis. Besides, we found that the db to m experimental mice compared to m to m control mice in 2 and 14 days sacrificed groups both reduce the gene and protein of FOXO1 in their kidneys. In summary, this study demonstrates that exosomes may be a novel mechanism to be involved in diabetic nephropathy, and abundant miR-669c-3p in exosomes may regulate the expression of FOXO1 in kidneys, and further give rise to renal fibrosis.