Transcriptomic signatures responding to PKM2 activator TEPP-46 in the hyperglycemic human renal proximal epithelial tubular cells

Pyruvate kinase M2 (PKM2), as the terminal and last rate-limiting enzyme of the glycolytic pathway, is an ideal enzyme for regulating metabolic phenotype. PKM2 tetramer activation has shown a protective role against diabetic kidney disease (DKD). However, the molecular mechanisms involved in diabetic tubular has not been investigated so far. In this study, we performed transcriptome gene expression profiling in human renal proximal tubular epithelial cell line (HK-2 cells) treated with high D-glucose (HG) for 7 days before the addition of 10-µM TEPP-46, an activator of PKM2 tetramerization, for a further 1 day in the presence of HG. Afterwards, we analyzed the differentially expressed (DE) genes and investigated gene relationships based on weighted gene co-expression network analysis (WGCNA). The results showed that 2,902 DE genes were identified (adjusted P-value = 0.05), where 2,509 DE genes (86.46%) were co-expressed in the key module. Four extremely down-regulated DE genes (HSPA8, HSPA2, HSPA1B and ARRB1) and three extremely up-regulated DE genes (GADD45A, IGFBP3 and SIAH1) enriched in the down-regulated endocytosis (hsa04144) and up-regulated p53 signaling pathway (hsa04115), respectively, were validated by the qRT-PCR experiments. The qRT-PCR results showed that the relative expression levels of HSPA8 (adjusted P-value = 4.45×10-34 & log2(FC) = -1.12), HSPA2 (adjusted P-value = 6.09×10-14 & log2(FC) = -1.27), HSPA1B (adjusted P-value = 1.14×10-11 & log2(FC) = -1.02) and ARRB1 (adjusted P-value = 2.60×10-5 & log2(FC) = -1.13) were significantly different (P-value < 0.05) from case group to control group. Furthermore, the interactions and predicted microRNAs of the key genes (HSPA8, HSPA2, HSPA1B and ARRB1) were visualized in networks. This study identified the key candidate transcriptomic biomarkers and biological pathways in the hyperglycemic HK-2 cells responding to the PKM2 activator TEPP-46. These results will be valuable for further research on PKM2 in DKD. Overall design: The obtained HK-2 cells were exposed to 25-mM HG medium as the hyperglycemic condition for 7 days. Thereafter, three replicates of cells exposed to HG were treated with (case group) or without (control group) 10-µM TEPP-46 for 1 day, separately. We extracted 2 µg RNA per cell replicate sample for RNA sample preparation, generated the sequencing libraries using NEBNext Library Prep Kit (NEB, USA) following the manufacturer's recommendations and sequenced on the Illumina Hiseq X ten platform to generate the 150 bp paired-end reads step by step.

丙酮酸激酶M2（Pyruvate kinase M2, PKM2）作为糖酵解通路的末端限速酶，是调控细胞代谢表型的理想靶点酶。PKM2四聚体激活已被证实对糖尿病肾病（Diabetic kidney disease, DKD）具有保护作用，但目前针对糖尿病性肾小管病变的分子机制尚未见相关研究报道。
本研究以人肾近端小管上皮细胞系（human renal proximal tubular epithelial cell line, HK-2细胞）为研究对象，先在高D-葡萄糖（high D-glucose, HG）培养条件中处理7天，随后加入10μM TEPP-46（一种PKM2四聚化激活剂）继续培养1天，全程维持高糖环境。后续通过转录组基因表达谱分析，基于加权基因共表达网络分析（Weighted Gene Co-expression Network Analysis, WGCNA）筛选差异表达（differentially expressed, DE）基因并探究基因间的调控关联。
研究结果显示，本研究共鉴定出2902个DE基因（校正P值=0.05），其中2509个（占比86.46%）DE基因在核心共表达模块中被富集。筛选得到4个极显著下调的DE基因（HSPA8、HSPA2、HSPA1B及ARRB1）与3个极显著上调的DE基因（GADD45A、IGFBP3及SIAH1），二者分别富集于内吞通路（hsa04144）与p53信号通路（hsa04115），并通过实时荧光定量PCR（quantitative real-time polymerase chain reaction, qRT-PCR）实验验证了上述基因的表达差异。
qRT-PCR结果表明，与对照组相比，实验组中HSPA8（校正P值=4.45×10⁻³⁴，log₂(FC)=-1.12）、HSPA2（校正P值=6.09×10⁻¹⁴，log₂(FC)=-1.27）、HSPA1B（校正P值=1.14×10⁻¹¹，log₂(FC)=-1.02）及ARRB1（校正P值=2.60×10⁻⁵，log₂(FC)=-1.13）的相对表达水平均存在显著统计学差异（P<0.05）。此外，本研究还对核心基因（HSPA8、HSPA2、HSPA1B及ARRB1）的相互作用关系与预测microRNA调控网络进行了可视化构建与展示。
本研究明确了高糖环境下HK-2细胞响应PKM2激活剂TEPP-46的关键候选转录组学生物标志物及生物学通路，相关成果可为后续PKM2在糖尿病肾病中的研究提供重要参考。
整体实验设计：将HK-2细胞置于含25mM高D-葡萄糖的培养基中培养7天，以构建高糖损伤模型。随后将高糖培养的细胞分为两组，每组设置3个生物学重复：实验组加入10μM TEPP-46继续培养1天，对照组不添加TEPP-46，同样培养1天。每个生物学重复样本提取2μg总RNA用于RNA样本制备，按照NEBNext文库制备试剂盒（NEB, 美国）的制造商操作指南构建测序文库，随后在Illumina Hiseq X Ten测序平台上进行测序，最终获得150bp双端测序读段。