Supplementary Material for: Renal ischemia induces ER-stress and impairs the reparative potency of scattered tubular-like cells

Background: CD24+/CD133+ scattered tubular-like cells (STCs) are surviving renal cells that acquire progenitor-like characteristics to repair other damaged kidney cells. Renal artery stenosis (RAS) impairs the reparative capacity of STCs, but the underlying mechanisms remain unknown. STCs contain abundant endoplasmic reticulum (ER), but its capacity to fold proteins could become saturated (ER-stress), leading to STC dysfunction. We hypothesized that RAS alters the expression of genes implicated in ER-stress in swine STCs. Methods: STCs were harvested from pig kidneys after 10 weeks of RAS or sham (n=6 each) and expression of ER-stress genes was assessed using mRNA-seq (n=3 each). To elucidate mechanisms regulating ER-stress genes in RAS-STCs, integrated mRNA-seq/microRNA (miRNA)-seq and transcription factor (TF) prediction analysis were performed. STC ER-stress was assessed in vitro using western blotting, serial block-face electron microscopy, and mass-spectrometry. The involvement of ER-stress in regulating the STC-protective effects was also assessed in vitro by their capacity to improve viability of injured human tubular epithelial cells. Results: RAS pigs developed significant renal dysfunction. mRNA-seq identified 25 ER-stress genes upregulated and 30 downregulated in RAS-STCs versus Normal-STCs. miRNAs were found to target over a third of all differentially expressed ER-stress genes, and almost half of genes encoding for the top 50 TFs involved in regulation of ER-stress genes were dysregulated in RAS-STCs. RAS-STCs exhibited higher ER-stress compared to Normal-STCs, reflected in significant ER dilation and formation of ER-mitochondria contacts, and increased levels of ER-stress-related amino acids. Importantly, ER-stress inhibition improved the reparative capacity of RAS-STCs in vitro. Conclusion: Renal ischemia alters expression of ER-stress-related genes in swine STCs, likely through post-transcriptional- and TF-regulatory mechanisms, which induces ER-stress and impairs their reparative potency. These alterations may limit the potential of STCs to repair damaged kidneys in subjects with RAS.