Fig. 1
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