Tubular epithelial cell-derived extracellular vesicles induce macrophage glycolysis by stabilizing HIF-1α in diabetic kidney disease.
Tubular epithelial cell-derived extracellular vesicles induce macrophage glycolysis by stabilizing HIF-1α in diabetic kidney disease.
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DOI：
10.1186/s10020-022-00525-1
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发表时间：
2022-08-12
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白蛋白尿是糖尿病肾病（DKD）的标志，可促进其进展，导致肾纤维化。肾脏巨噬细胞功能复杂，受巨噬细胞代谢状态的影响。然而，糖尿病肾巨噬细胞的代谢状态和白蛋白尿对巨噬细胞代谢状态的影响知之甚少。采用透射电镜、纳米颗粒示踪分析和蛋白质印迹法对肾小管上皮细胞（HK-2）的细胞外囊泡（EV）进行了评价。使用RT-qPCR和蛋白质印迹法检测与HSA处理的HK-2细胞衍生的EV共培养的巨噬细胞中糖酵解酶的表达。在HIF-1α siRNA预转染的巨噬细胞与HSA处理的HK-2细胞来源的EV共培养中探索EV相关的HIF-1α在糖酵解介导中的潜在作用，并使用蛋白质印迹法测量HIF-1α羟基化的程度。此外，我们通过尾静脉给db/db小鼠注射EV，每周两次，持续4周。使用CD 11b微珠分离肾巨噬细胞，并应用荧光法确认这些巨噬细胞中糖酵解酶和HIF-1α的水平。糖尿病肾巨噬细胞与HSA处理的HK-2细胞共培养后，糖酵解被激活。此外，HSA处理的HK-2细胞衍生的EV在体内和体外均促进巨噬细胞糖酵解。使用糖酵解抑制剂2-DG抑制巨噬细胞中的糖酵解激活降低了炎症和纤维化基因的表达。从机制上讲，发现来自HSA刺激的HK-2细胞的EV通过稳定HIF-1α来加速巨噬细胞糖酵解。我们还发现，在HSA处理的HK-2细胞衍生的EV中，几种已报道稳定HIF-1α表达的miRNA和lncRNA增加。我们的研究表明，白蛋白尿通过稳定HIF-1α通过肾小管上皮细胞来源的EV诱导肾脏巨噬细胞糖酵解，表明调节巨噬细胞糖酵解可能为DKD患者，特别是大量白蛋白尿患者提供新的治疗策略。在线版本包含补充材料，可通过10.1186/s10020-022-00525-1获得。
Albuminuria is a hallmark of diabetic kidney disease (DKD) that promotes its progression, leading to renal fibrosis. Renal macrophage function is complex and influenced by macrophage metabolic status. However, the metabolic state of diabetic renal macrophages and the impact of albuminuria on the macrophage metabolic state are poorly understood. Extracellular vesicles (EVs) from tubular epithelial cells (HK-2) were evaluated using transmission electron microscopy, nanoparticle tracking analysis and western blotting. Glycolytic enzyme expression in macrophages co-cultured with HSA-treated HK-2 cell-derived EVs was detected using RT-qPCR and western blotting. The potential role of EV-associated HIF-1α in the mediation of glycolysis was explored in HIF-1α siRNA pre-transfected macrophages co-cultured with HSA-treated HK-2 cell-derived EVs, and the extent of HIF-1α hydroxylation was measured using western blotting. Additionally, we injected db/db mice with EVs via the caudal vein twice a week for 4 weeks. Renal macrophages were isolated using CD11b microbeads, and immunohistofluorescence was applied to confirm the levels of glycolytic enzymes and HIF-1α in these macrophages. Glycolysis was activated in diabetic renal macrophages after co-culture with HSA-treated HK-2 cells. Moreover, HSA-treated HK-2 cell-derived EVs promoted macrophage glycolysis both in vivo and in vitro. Inhibition of glycolysis activation in macrophages using the glycolysis inhibitor 2-DG decreased the expression of both inflammatory and fibrotic genes. Mechanistically, EVs from HSA-stimulated HK-2 cells were found to accelerate macrophage glycolysis by stabilizing HIF-1α. We also found that several miRNAs and lncRNAs, which have been reported to stabilize HIF-1α expression, were increased in HSA-treated HK-2 cell-derived EVs. Our study suggested that albuminuria induced renal macrophage glycolysis through tubular epithelial cell-derived EVs by stabilizing HIF-1α, indicating that regulation of macrophage glycolysis may offer a new treatment strategy for DKD patients, especially those with macroalbuminuria. The online version contains supplementary material available at 10.1186/s10020-022-00525-1.
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