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Transcriptome-based analysis of molecular pathways for clusterin functions in kidney cells Dairi, Ghida Saleh


Background: Clusterin (CLU) is a chaperone-like protein. Our previous studies have demonstrated that CLU protects kidney from ischemia-reperfusion injury (IRI) and enhances renal repair after IRI; however, the molecular pathways for its functions in the kidney are not fully understood. This study was designed to investigate CLU-mediating pathways in kidney cells by using bioinformatics analysis. Materials and Methods: An in vitro model of kidney tissue using CLU null renal tubular epithelial cells (TECs) was established for this research project. An immortalized CLU null TEC cell line was generated from a CLU knockout (KO) mouse, and was stably expressing pHEX6300 plasmid containg human CLU cDNA (TEC-CLUhCLU), so that this cell line constitutively expresses human CLU protein, whereas control cell line (TEC-CLU-/-) was generated from the same parental CLU null TEC cell line by expressing empty pHEX6300. Both TEC-CLUhCLU and TEC-CLU-/- cell lines were exposed to either normoxia or hypoxia (1% O2). Transcriptome profiling with a significant 2-fold change (FC) (FC ≥ 2, p ≤ 0.05) was performed using SurePrint G3 Mouse Gene Expression 8×60K microarray, and the signaling pathways was ranked by using Ingenuity pathway analysis (IPA). Results: Here, we showed that compared to CLU null TEC-CLU-/- controls ectopic expression of human CLU in CLU null kidney cells (TEC-CLUhCLU) promoted cell growth but inhibited migration in normoxia, and enhanced cell survival in hypoxia. CLU affected expression of 3864 transcripts (1893 up-regulated) in normoxia and 3670 transcripts (1925 up-regulated) in hypoxia. CLU functions including cell proliferation, survival and adhesion in normoxia were associated mostly with AKT2 dependent PI3K/AKT, PTEN, VEGF and ERK/MAPK signaling and as well with GSK3B-mediated cell cycle progression. In addition to unfolded protein response (UPR) and/or endoplasmic reticulum (ER) stress, CLU-enhanced cell survival in hypoxia was also associated with Foxo3/PIK3CD/MAPK1-dependent PI3K/AKT, HIF-α, PTEN, VEGF and ERK/MAPK signaling. Conclusion: Our data showed that CLU functions in kidney cells were mediated in a cascade manner mainly by PI3K/AKT, PTEN, VEGF and ERK/MAPK signaling, and specifically by activation of UPR/ER stress in hypoxia, providing new insights into the protective role of CLU in the kidney.

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