糖尿病肾周脂肪细胞来源小细胞外囊泡促进肾小管上皮细胞上皮-间质转化

Diabetic perirenal adipocyte-derived small extracellular vesicles promote epithelial-mesenchymal transition in renal tubular epithelial cells

目的探讨糖尿病状态下肾周脂肪组织(PRAT)脂肪细胞来源小细胞外囊泡(sEVs)对肾小管上皮细胞的生物行为影响以及其分子机制。方法提取原代db/m、db/db小鼠PRAT脂肪细胞进行体外培养, 收集培养上清并通过超速离心法提取sEVs(NDM-sEVs^(PRAT-Adipo), DM-sEVs^(PRAT-Adipo))。将sEVs与人肾小管上皮细胞株(HK-2)细胞孵育, 观察其增殖、凋亡、自噬以及上皮-间质转化(EMT)水平。通过质谱技术分析sEVs蛋白成分, 探讨其作用的分子机制。结果 CCK8结果表明DM-sEVs^(PRAT-Adipo)干预后HK-2细胞的增殖水平与2个对照组(Ctrl组和NDM-sEVs^(PRAT-Adipo)干预组)相比无明显改变。Western印迹法结果表明, 对比2个对照组, DM-sEVs^(PRAT-Adipo)干预组的凋亡(Bcl-2、Cleaved-caspase 3、Caspase 3)和自噬(p62、LC3BⅠ、LC3BⅡ)水平无明显改变。Western印迹法和免疫荧光结果表明, 与2个对照组比较, DM-sEVs^(PRAT-Adipo)干预可上调HK-2的间质细胞标志蛋白(Vimentin、α-SMA、Snail2)表达水平, 下调上皮细胞标志蛋白ZO-1表达水平。sEVs质谱分析结果表明, DM-sEVs^(PRAT-Adipo)与NDM-sEVs^(PRAT-Adipo)的差异蛋白富集于EMT相关通路。其中, DM-sEVs^(PRAT-Adipo)富集血小板反应蛋白(THBS1), 该蛋白与EMT密切相关。结论糖尿病状态下, PRAT来源脂肪细胞分泌sEVs促进肾小管上皮细胞EMT上调, 此过程可能由sEVs中所富集的THBS1介导。

Objective:To investigate the impact of small extracellular vesicles(sEVs)derived from perirenal adipose cells on the biological behavior of renal tubular epithelial cells under diabetic conditions and the underlying molecular mechanisms.Methods:Primary perirenal adipose cells were extracted from db/m and db/db mice for in vitro culture.The culture supernatant was collected and sEVs(NDM-sEVs ^(PRAT-Adipo),DM-sEVs ^(PRAT-Adipo))were extracted by ultracentrifugation.The sEVs were incubated with human renal tubular epithelial cell line(HK-2)to observe changes in their proliferation,apoptosis,autophagy,and epithelial-mesenchymal transition(EMT)levels.The protein composition of sEVs was analyzed using mass spectrometry to explore the molecular mechanisms.Results:CCK8 results showed that the proliferation level of HK-2 cells after DM-sEVs ^(PRAT-Adipo) intervention did not change significantly compared with the two control groups(Ctrl group and NDM-sEVs ^(PRAT-Adipo) intervention group).Western Blot(WB)results indicated that there were no significant changes in apoptosis levels(Bcl-2,Cleaved-caspase 3,Caspase 3)and autophagy levels(p62,LC3BⅠ,LC3BⅡ)in the DM-sEVs ^(PRAT-Adipo) intervention group compared with the two control groups.WB and immunofluorescence results demonstrated that DM-sEVs ^(PRAT-Adipo) intervention upregulated the expression levels of mesenchymal cell marker proteins(Vimentin,α-SMA,Snail2)and downregulated the expression level of epithelial cell marker protein ZO-1 in HK-2 cells compared with the two control groups.Mass spectrometry analysis of sEVs revealed that the differential proteins between DM-sEVs ^(PRAT-Adipo) and NDM-sEVs ^(PRAT-Adipo) were enriched in EMT-related pathways.Among them,the enrichment of thrombospondin(THBS1)in DM-sEVs ^(PRAT-Adipo) might be involved in the regulation of EMT in HK-2 cells.Conclusion:Under diabetic conditions,sEVs secreted by PRAT-derived adipocytes promote the upregulation of EMT in renal tubular epithelial cells,a process that may be mediated by the enrichment of THBS1 in sEVs.