Transcriptome analysis and weighted gene co-expression network reveal candidate genes and pathways responses to lactate dehydrogenase inhibition (oxamate) in hyperglycemic human renal proximal epithelial tubular cells. Transcriptome analysis and weighted gene co-expression network reveal candidate genes and pathways responses to lactate dehydrogenase inhibition (oxamate) in hyperglycemic human renal proximal epithelial tubular cells

Diabetic kidney disease (DKD) is the leading cause of both chronic kidney disease (CKD) and end-stage renal disease (ESRD). In this study, we performed transcriptome gene expression profiling of kidney tissues in human renal proximal epithelial tubular cell line (HK-2) treated with high D-glucose (HG) for 7 days before the addition of 40 mM oxamate for a further 24 hours 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). Accordingly, enrichment analyses of GO terms and KEGG pathways showed that several pathways (e.g., lysosome (hsa04142) and p53 signaling pathway (hsa04115)) may be involved in a response of HK-2 cells to oxamate. Moreover, via WGCNA, we identified two modules: both the turquoise and blue modules were enriched in pathways associated with lysosome. However, the P53 signaling pathway was only found using all 3,884 DE genes. Furthermore, the key hub genes IGFBP3 interacted with 6 up-regulated and 12 down-regulated DE genes in the network that were enriched in the P53 signaling pathway. This is the first study reporting co-expression patterns of a gene network after lactate dehydrogenase inhibition in HK-2 cells. Our results may contribute to our understanding of the underlying molecular mechanism of in vitro reprogramming under hyperglycemic stress that orchestrates the survival and functions of HK-2 cells. Overall design: Six replicates of HK-2 cells were firstly exposed to HG treatments for seven days, then three replicates of them were exposed 40 mM oxamate (case group), while the other three replicates were still under the same HG condition but 40 mM oxamate expose (control group). Unfortunately, one replicate of case group failed in RNA sample preparation, so five replicates of cells were finally chosen for RNA sequencing in this study.

糖尿病肾病（Diabetic kidney disease, DKD）是慢性肾脏病（Chronic kidney disease, CKD）与终末期肾病（End-stage renal disease, ESRD）的首要致病诱因。本研究针对人类肾近端上皮管状细胞系（HK-2）开展转录组基因表达谱分析：先以高浓度D-葡萄糖（HG）处理细胞7天，随后在持续高糖环境中加入40 mM草氨酸盐继续处理24小时。之后，我们分析了差异表达（DE）基因，并基于加权基因共表达网络分析（WGCNA）探究了基因间的关联关系。相应地，基因本体（GO）术语富集分析与京都基因与基因组百科全书（KEGG）通路富集分析结果显示，溶酶体（hsa04142）、p53信号通路（hsa04115）等多条通路可能参与HK-2细胞对草氨酸盐的应答反应。此外，通过WGCNA我们鉴定出两个模块：绿松石模块与蓝色模块均富集于溶酶体相关通路，但p53信号通路仅在全部3884个DE基因的分析中被检出。进一步研究发现，核心枢纽基因IGFBP3可与该网络中6个上调、12个下调的DE基因发生相互作用，这些基因均富集于p53信号通路。本研究首次报道了HK-2细胞在乳酸脱氢酶抑制后的基因共表达模式。我们的研究结果有助于阐明高血糖应激下HK-2细胞存活与功能调控的体外重编程潜在分子机制。整体实验设计：首先将6组重复的HK-2细胞置于高糖环境中培养7天，随后将其中3组细胞加入40 mM草氨酸盐处理（实验组），剩余3组仍维持高糖环境但不加入草氨酸盐（对照组）。遗憾的是，实验组中有1组样本的RNA制备失败，因此本研究最终选取5组细胞样本进行RNA测序。