T细胞来源微囊泡介导miR-186调控足细胞损伤的机制研究

Podocyte injury has emerged as a central path-mechanism underlying focal segmental glomerular sclerosis (FSGS). Recently, circulation factor is regarded as a main pathogenic factor of FSGS. Our previous study screened for differentially expressed plasma miRNAs in patients with FSGS with nephrotic proteinuria relative to healthy controls using miRNA PCR arrays and quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis to validated candidate plasma miRNAs. The result indicated that plasma miR-186 may be a biomarker for FSGS with nephrotic proteinuria. Microvesicles (MVs), also known as extracellular vesicles, are small (30–1000 nm) membrane-bound particles released from eukaryotic cells and play a pivotal role in mediating cell-to-cell communication. We found that miR-186 was high expression in CD3+T cells and the MVs derived from CD3+T cells (T-MVs) in the patients with FSGS. Moreover, these T-MVs could induce proteinuria in BalB/C mice. So we hypothesized that MVs secreted from T cells might contribute to the miR-186 promote podocytes injure. In this proposed project, we will conduct the clinical cohort to analysis the correlation with cell percentages of T lymphocyte subset and plasma miR-186. Through the use of cultured T cells and transfect miR-186 mimics to map T-MVs production in vitro. We next explore the potential pathogenic mechanism of miR-186 in podocytes cytoskeleton, cell migration and apoptosis via its target gene including CDC42，CD2AP and ROCK1.To further confirm that miR-186 derived from T-MVs would contribute the process of the FSGS as “circulation factor”. The project will highlight the mechanism of podocytes injured in FSGS and provide the new therapy target.

足细胞损伤是局灶节段性肾小球硬化(FSGS)发生发展的关键环节。课题组前期利用生物信息学分析及临床队列验证发现血浆miR-186水平与FSGS患者蛋白尿水平及疾病活动呈正相关。并发现FSGS患者外周CD3+T细胞内及其所分泌的微囊泡（Microvesicles，MVs）中miR-186水平都显著上调；进一步将过表达miR-186的T细胞所分泌的MVs注射小鼠体内，发现小鼠出现蛋白尿。提示T细胞分泌的MVs可通过介导miR-186诱导足细胞损伤的作用。本项目拟进一步通过临床队列和体外细胞实验证实T细胞为血浆miR-186的重要来源，并明确分泌miR-186的T细胞亚群；通过体内外实验，探讨T细胞是否通过上调miR-186的表达及分泌水平诱导足细胞损伤，并明确诱导足细胞损伤的分子机制；研究结果有望发现FSGS新的致病机制及干预靶点，为临床治疗提供新的思路。

局灶节段性肾小球硬化(FSGS)是演变成CKD的重要病因。足细胞损伤是FSGS发生发展的关键环节，是导致蛋白尿、肾小球硬化及肾功能进行性恶化的重要病理基础，但引起足细胞损伤的分子基础及机制尚不十分明确。近二十年来研究发现，FSGS患者血浆中存在某种可诱导足细胞或肾小球损伤的物质，即“循环因子”，是FSGS主要病理机制之一。鉴定该“循环因子”一直是该领域热点课题。课题组前期利用生物信息学分析及临床队列验证发现血浆miR-186水平与FSGS患者蛋白尿水平及疾病活动呈正相关。并发现FSGS患者外周CD3+T细胞内及其所分泌的微囊泡（Microvesicles，MVs）中miR-186水平都显著上调；进一步将过表达miR-186的T细胞所分泌的MVs注射小鼠体内，发现小鼠出现蛋白尿。提示T细胞分泌的MVs可通过介导miR-186诱导足细胞损伤的作用。本项目进一步通过临床队列和体内外实验证实CD8+T淋巴细胞的数量与miR-186水平呈明显正相关，而且CD8+T淋巴细胞表达miR-186的水平要明显高于其他血细胞，提示CD8+T淋巴细胞有可能为血浆miR-186的主要来源细胞。通过分离血浆外泌体并对miR-186检测发现，血浆中miR-186主要以外泌体为载体的形式稳定存在。通过CD8+T细胞与足细胞共培养实验发现，TMV-miR186可明显被足细胞所摄取并上调足细胞的凋亡水平以及骨架紊乱程度。而且足细胞过表达miR-186后，可抑制关键骨架蛋白基因（CD2AP、CDC42及ROCK1蛋白）的翻译水平。为探寻外源性miRNA能否进入足细胞并发挥类似于内源性miRNA的功能，我们通过尾静脉注射小鼠，发现TMV-miR186可在肾组织中大量聚集，原位杂交、电镜和免疫荧光结果显示该miRNA可被足细胞所摄取，并导致小鼠产生尿蛋白，经肾组织超微结构观察发现，注射TMV-miR186的小鼠，部分肾小球足细胞足突节段融合。研究结果有望发现FSGS新的致病机制及干预靶点，为临床治疗提供新的思路。