Fig 2: MiR-381-3p inhibited NFAT5 by directly targeting its 3’UTR.A, B DEGs between NC and Pi group were presented by heatmap and volcano plot. C The intersection of different online databases and DEGs was presented by Venn graph. D The binding sites of miR-381-3p and NFAT5 were predicted by TargetScan. E The combination of miR-381-3p and NFAT5 was verified by dual-luciferase reporter gene assay, *P < 0.05. F, G The protein and mRNA expression levels of NFAT5 in HA-VSMCs were detected after transfection of miR-381-3p mimics, *P < 0.05 compared with the mimics NC group; #P < 0.05 compared with the Pi + mimics NC group; &P < 0.05 compared with the Pi + mimics 50 nM group. H, I The protein and mRNA expression levels of NFAT5 in HA-VSMCs were detected under treatment of different exosomes, *P < 0.05 compared with the NC group; #P < 0.05 compared with the Pi group; $P < 0.05 compared with the Pi + Exo group.

Fig 3: Circ_0010235 silencing augmented the anticancer activity of 2‐MeOE2 in vivo. (a) Tumor volume was monitored. (b) Tumor weight was examined after 23 days inoculation. (c, d) The expression levels of circ_0010235 and miR‐34a‐5p were measured by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) in resected xenograft tumor tissues. (e) The protein levels of NFAT5 were measured by western blot assay in resected xenograft tumor tissues. (f) The expression levels of NFAT5, Ki67 and MMP2 in the collected xenograft tumors were estimated by immunohistochemistry (IHC) assay. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Fig 4: Hyperosmolality-induced upregulation of Ranbp3l is abrogated in NFAT5 deficient cells. A RNA-Seq analyses show the expression of Ranbp3l and other Ranbp family members in primary murine IMCD-cells cultivated at 300 or 600 msomol/kg displayed as heatmap in FPKM (Fragments per kilo base per million mapped reads), (n = 2). Blue to red represents lower to higher expression. B Pearson correlation analysis of genes affected by hyperosmolality (log2 fold change > 1, <− 1) between the mpkCCD cell line and primary cultivated IMCD cells. (n = 2–4), blue = overlapping upregulated genes, red = overlapping down regulated genes. C WT (wildtype) mpKCCD cells were cultivated under isosmotic and hyperosmotic conditions for 7d and subjected to immunofluorescence staining with anti-NFAT5 antibody and DAPI for nucleus visualization. Scale bar: 100 μm (D) Western blot analysis of NFAT5 expression in scramble (Scr) and NFAT5 deficient single cell clones N1 and N3. The expression of GAPDH served as loading control. E The relative expression of Ranbp3l, Aqp2 and Slc6a12 in Scr vs. NFAT5-deficient clones was analyzed by RT-qPCR in cells cultivated under hyperosmotic conditions (n = 3). Values represent mean ± SEM (error bars). *, p < 0.05; **, p < 0.01, 1-way ANOVA. F Efficient in vitro deletion of NAFT5 in primary cultured IMCD cells using Nfat5flx/fl-Ubc-Cre-ERT2+/− mice. Representative immunofluorescence of control and 4-OH-TM treated cells for NFAT5 under hyperosmolar cell cultivation. Scale bar: 100 μm (G) FPKM values of all known kidney-specific NFAT5 targets, including Ranbp3l for control and 4-OH-TM treated cells at 300 mosmol/kg and 600 mosmol/kg (n = 2). (H) Induction of promotor activity under hyperosmotic conditions with the Ranbp3l-2 kb construct. (n = 4, each with duplicates). **, p < 0.01, Student’s t test (I) Luciferase assay with different Ranbp3l promoter fragments. Promoter activity is represented relative to the Ranbp3l-2 kb construct. (n = 4, with two technical replicates) n.s. > 0.05, *, p < 0.05, 1way ANOVA

Fig 5: RNA-seq for potential target of TonEBP. (A) Volcano plot of DEGs between the sh-NC group and sh-TonEBP group. p-value < 0.05 and |(log2FoldChange)| > 1. (B) GO enrichment scatter plot. (C) KEGG pathway enrichment analysis plot. (D) GO enrichment chord diagram and bar plot legend for GO terms. (E) Wayne diagram showing upregulated DEGs in LPS-stimulated BV2 cells and downregulated DEGs in TonEBP-deficient BV2 cells. (F,G) Immunohistochemistry (scale bar = 50 μm) (F) and IHC score (G) for PELI1 in brain tissues of humans in control group and ICH group. N = 3. The data are presented as mean ± SD. ** p < 0.01.