NFAT5 and HIF-1α are independently up-regulated by hypoxia.

<p>HEK293 cells cultured at 300 mosmol were transfected with control (C), NFAT5 or HIF-1α siRNA. 48 hrs after transfection the cells were cultured in normoxia (N) or 8 hrs of hypoxia (H). <b>A.</b> Protein abundance of NFAT5 and HIF-1α were studied by Western blot in cells transfected with siRNA against NFAT5. <b>B.</b> Protein abundance of NFAT5 and HIF-1α were studied by Western blot in cells transfected with siRNA against HIF-1α. A representative picture is shown in the upper section. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.</p

Experimental I/R induced renal Aldose Reductase (AR) expression.

<p><b>A.</b> AR protein abundance in protein homogenates from cortex and medulla of rat kidney determined by Western blot. A representative picture is shown in the upper section. <b>B.</b> AR mRNA abundance in kidney cortex and medulla measured by qRT-PCR. Bar graph represents Mean ± SEM. * or & indicates P<0.05; n = 5 (*<i>vs</i> sham medulla & <i>vs</i> sham cortex).</p

NFAT5 has a protective role against hypoxia.

<p>HEK293 cells cultured at 300 mosmol were transfected with control and NFAT5 siRNA. 48 hrs after transfection the cells were exposed for 8 hrs to 2.5% PO₂. <b>A.</b> Western blot of NFAT5. <b>B.</b> LDH activity was assayed in cell culture media and cell lysate by spectrometric determination of NADH. <b>C.</b> Western blot of M30. <b>D.</b> Western blot of Cleaved caspase-3. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.</p

Hypoxia induces NFAT5 in cell culture.

<p>A. HEK293 cells cultured at 300 mosmol were subjected to dose-response curve of hypoxia (21, 5, 2.5 and 1% O₂). NFAT5 (A1) and HIF-1α (A2) protein abundance was determinate by Western blot. B. Using 2.5% of PO₂, cells were exposed for 0, 4, 8, and 16 hrs to analyse the HIF-1α gene expression by qRT-PCR (B1) and Western blot (B3). NFAT5 gene expression was also determined by qRT-PCR (B2) and Western blot (B4). Protein abundance and mRNA were normalized by tubulin (Tub) and 18S, respectively. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.</p

Wortmannin inhibited the NFAT5 activation by hypertonicity and hypoxia in HEK293 cells.

<p><b>A.</b> Cultures at 300 mosmol were incubated with DMSO (D) or Wortmannin (W) by 1 hour. Then the cells were cultured in normoxia (N) or 8 hrs of hypoxia (H) and NFAT5 abundance was studied by Western blot. <b>B.</b> Cells cultured at 300 mosmol were transfected with HRE-Luciferase and 24 hrs after transfection the cells were incubated with DMSO (D) or Wortmannin (W) by 1 hour. After this treatment, the cells were cultured in normoxia (300 or 500 mOsM) or 8 hrs of hypoxia (300 or 500 mOsM) and the luciferase activity was assayed. <b>C.</b> Cells cultured at 300 mosmol were cotransfected with HRE-Luciferase and siRNA (control or NFAT5). 48 hrs after transfection the cells were cultured in normoxia or 8 hrs of hypoxia (300 or 500 mOsM) and the luciferase activity was assayed. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.</p

Anoxia increases NFAT5 protein abundance and promotes nuclear translocation.

<p><b>A.</b> Rat primary IMCD cells in isotonic (300 mOsM) or hypertonic (640 mOsM) medium were exposed to anoxia (replacement of O₂ by N₂) for 0, 8, or 16 hrs. We prepared total protein homogenates and determined NFAT5 protein abundance by Western blot. A representative picture is shown in the upper section and the graph shows mean ± SEM. * or & P≤0.05; n = 5. (*<i>vs.</i> 300 mosmol/normoxia and & <i>vs.</i> 640 mosmol/normoxia). <b>B.</b> NFAT5 cellular distribution after 2 hrs of anoxia evaluated in primary IMCD cells by immunofluorescence. Green = NFAT5 labelling (Alexa488); blue = nuclei (Hoechst 33258). <b>C.</b> HEK293 cells stably expressing ORE-X cultured at 300 mosmol or 500 mosmol by 16 hrs; during this time the cells were exposed for 0, 8 or 16 hrs to anoxia, and luciferase reporter assay was used to evaluate transcription activity; Bar graph represents Mean ± SEM. (* or &, P<0.05; n = 5). <b>D.</b> HEK293 cells cultured at 300 mosmol were exposed by 2 hrs to anoxia (a) or normoxia (n). Nuclear and cytoplasmatic fractions were separated by NE-PER and NFAT5 abundance was determined by Western blot. Bar graph represents Mean ± SEM. (* P<0.05; n = 5).</p

NFAT5-regulators protein, ATM and PI3K, were induced in post-ischemic kidneys.

<p>ATM and PI3K (p110α) protein abundance was measured by Western blot in cortex or medulla from rat kidney. A. A representative picture is shown in the upper section (n = 5). <b>B.</b> Relative ATM abundance. <b>C.</b> Relative p110α abundance. Bar graph represents Mean ± SEM. * or & indicates P<0.05; n = 5 (* <i>vs</i> sham medulla & <i>vs</i> sham cortex).</p

Kidney function after experimental I/R.

<p><b>A.</b> Tissue damage evaluated by PAS staining. Brush border, epithelial flattening and mitosis were present in kidneys from I/R animals (arrows). <b>B.</b> Serum creatinine (mg/dl) of sham and 24–96 hrs post-ischemia. <b>C.</b> Urine and plasma ratio (U/P) of osmolality of sham and 24–96 hrs post-ischemia. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.</p