Transcriptomic profiling of tight junctions dysfunction in endothelial cells and epithelial cells in renal failure
Authors: Xu Jen , Ho Lilun , Wu Chiachun , Lim Kenneth , Lu Tzongshi 
Published: November 2023

BackgroundChronic kidney disease (CKD) has been recently identified as a significant risk factor for vascular diseases such as cerebral small‐vessel diseases (CSVD), and up to 50% of deaths in patients with end‐stage renal failure are due to cardiovascular disease (CVD). There is a 10 to 30‐fold higher cardiovascular mortality in CKD patients than in the age‐matched general population, despite adjustment for classical cardiovascular risk factors. Tight junctions (TJ) are specialized membrane domains that play multiple functions in kidney epithelial cells, including the maintenance of cellular polarity and work as a primary regulatory barrier particularly in cerebral microvasculatures and kideny. Studies indicate emerging links between TJ dysfunction and the development of kidney disease and following CVD and CSVD. The goal of this study was to elucidate the transcription profiling related to TJ dysfunction and its role in preventing renal failure through the regulation of TJs.MethodsTo profile the exhaustive signatures in TJ dysfunction human endothelium and epithelium, we performed whole transcriptome analysis to explore novel genes in primary human aortic endothelial cells (HAECs), human brain microvascular endothelial cells (HBMECs) and immortalized human kidney proximal tubule epithelial cells (HK‐2) using our CKD models, in vitro. In addition, we analyzed human arteries from CVD and CKD patients. Total RNA was extracted using the Nucleospin RNA isolation kit (Clontech). cDNA was synthesized and fragmented using SMART‐seq2 and Nextera XT (Illumina), respectively to generate libraries for sequencing on Hi‐seq 2000 (Illumina). The abundancies of each gene, (FPKM, fragment per kilo‐base exon per million mapped reads), were estimated using RNA‐seq by Expectation Maximization (RSEM). Log 2 transformed FPKM were normalized by quantile normalization, which were then subjected to differential expression analysis to calculate significant fold change (Gene‐E, Broad Institute). Gene selections were performed by the combinations of fold‐changes on log 2 ratio, and p value < 0.05. Data were analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) and The Database for Annotation, Visualization and Integrated Discovery v6.7 (DAVID, http://david.abcc.ncifcrf.gov/).ResultsPathway analysis among the differentially expressed genes in comparing normal samples to CKD groups revealed that these genes are mainly involved in cytoskeleton formation, cell metabolic, proliferation, and apoptosis processes. Among these TJ dysfunction related genes, heat shock protein 70 families (HSPA1, HSPB7), SYT, SNAP25, and aryl hydrocarbon receptor nuclear translocator (ARNT) in HIF‐1signaling pathways were identified. In addition, MT‐RNR1 and cannabinoid receptor 1 were also detected that are involved in ATP production in the mitochondria and as co‐chaperone for HSP70.ConclusionWe have identified a group of genes differentially expressed in healthy and CKD endothelial cells and epithelial cells. Our data contribute to our understanding of the direct effects in the presence of TJ dysfunction at endothelial cells and epithelial cells. This gene set may have pathophysiological significance and may serve to inform rational development of therapeutic strategies to prevent CVD adverse effects in CKD patients.