Bioinformatics analysis of proteomics profiles in senescent human primary proximal tubule epithelial cells.

Dysfunction of renal tubule epithelial cells is associated with renal tubulointerstitial fibrosis. Exploration of the proteomic profiles of senesced tubule epithelial cells is essential to elucidate the mechanism of tubulointerstitium development. Primary human proximal tubule epithelial cells from passage 3 (P3) and passage 6 (P6) were selected for evaluation. EdU and SA-β-galactosidase staining were used to detect cell senescence. p53, p21, and p16 were detected by Western blot analysis. Liquid chromatography mass spectrometry (LC-MS) was used to examine differentially expressed proteins (DEPs) between P6 and P3 cells. The expression of DEPs was examined by Western blot analysis. Bioinformatics analysis was performed by protein-protein interaction and gene ontology analyses. The majority of tubule cells from passage 6 (P6) stained positive for SA-β-galactosidase, whereas passage 3 (P3) cells were negative. Senescence biomarkers, including p53, p21, and p16, were upregulated in P6 cells relative to P3 cells. EdU staining results showed a lower rate of EdU positive cells in P6 cells than in P3 cells. LC-MS was used to examine DEPs between P6 and P3 cells. These DEPs are involved in glycolysis, response to stress, cytoskeleton regulation, oxidative reduction, ATP binding, and oxidative stress. Using Western blot analysis, we validated the down-regulation of AKR1B1, EEF2, EEF1A1, and HSP90 and the up-regulation of VIM in P6 cells seen in the LC-MS data. More importantly, we built the molecular network based on biological functions and protein-protein interactions and found that the DEPs are involved in translation elongation, stress, and glycolysis, and that they are all associated with cytoskeleton regulation, which regulates senescent cell activities such as apoptosis and EMT in tubule epithelial cells. We explored proteomic profile changes in cell culture-induced senescent cells and built senescence-associated molecular networks, which will help to elucidate the mechanisms of senescence in human proximal tubule epithelial cells.

肾小管上皮细胞功能障碍与肾小管间质纤维化有关。探究衰老肾小管上皮细胞的蛋白质组学特征对于阐明肾小管间质发展的机制至关重要。 选取第3代（P3）和第6代（P6）的原代人近端肾小管上皮细胞进行评估。采用EdU和SA -β -半乳糖苷酶染色检测细胞衰老。通过蛋白质印迹分析检测p53、p21和p16。利用液相色谱 - 质谱（LC - MS）检测P6和P3细胞之间的差异表达蛋白（DEPs）。通过蛋白质印迹分析检测DEPs的表达。通过蛋白质 - 蛋白质相互作用和基因本体分析进行生物信息学分析。 第6代（P6）的大多数肾小管细胞SA -β -半乳糖苷酶染色呈阳性，而第3代（P3）细胞为阴性。与P3细胞相比，P6细胞中的衰老生物标志物，包括p53、p21和p16，表达上调。EdU染色结果显示，P6细胞中EdU阳性细胞的比例低于P3细胞。利用LC - MS检测P6和P3细胞之间的DEPs。这些DEPs参与糖酵解、应激反应、细胞骨架调节、氧化还原、ATP结合和氧化应激。通过蛋白质印迹分析，我们验证了在LC - MS数据中所见的P6细胞中AKR1B1、EEF2、EEF1A1和HSP90的下调以及VIM的上调。更重要的是，我们基于生物学功能和蛋白质 - 蛋白质相互作用构建了分子网络，发现DEPs参与翻译延伸、应激和糖酵解，并且它们都与细胞骨架调节有关，而细胞骨架调节可调控肾小管上皮细胞中的衰老细胞活动，如凋亡和上皮 - 间质转化（EMT）。 我们探究了细胞培养诱导的衰老细胞中蛋白质组学特征的变化，并构建了衰老相关的分子网络，这将有助于阐明人近端肾小管上皮细胞的衰老机制。