NFAT5 is activated by hypoxia: role in ischemia and reperfusion in the rat kidney

Abstract

The current hypothesis postulates that NFAT5 activation in the kidney's inner medulla is due to hypertonicity, resulting in cell protection. Additionally, the renal medulla is hypoxic (10-18 mmHg); however there is no information about the effect of hypoxia on NFAT5. Using in vivo and in vitro models, we evaluated the effect of reducing the partial pressure of oxygen (PO(2)) on NFAT5 activity. We found that 1) Anoxia increased NFAT5 expression and nuclear translocation in primary cultures of IMCD cells from rat kidney. 2) Anoxia increased transcriptional activity and nuclear translocation of NFAT5 in HEK293 cells. 3) The dose-response curve demonstrated that HIF-1α peaked at 2.5% and NFAT5 at 1% of O(2). 4) At 2.5% of O(2), the time-course curve of hypoxia demonstrated earlier induction of HIF-1α gene expression than NFAT5. 5) siRNA knockdown of NFAT5 increased the hypoxia-induced cell death. 6) siRNA knockdown of HIF-1α did not affect the NFAT5 induction by hypoxia. Additionally, HIF-1α was still induced by hypoxia even when NFAT5 was knocked down. 7) NFAT5 and HIF-1α expression were increased in kidney (cortex and medulla) from rats subjected to an experimental model of ischemia and reperfusion (I/R). 7) Experimental I/R increased the NFAT5-target gene aldose reductase (AR). 8) NFAT5 activators (ATM and PI3K) were induced in vitro (HEK293 cells) and in vivo (I/R kidneys) with the same timing of NFAT5. 8) Wortmannin, which inhibits ATM and PI3K, reduces hypoxia-induced NFAT5 transcriptional activation in HEK293 cells. These results demonstrate for the first time that NFAT5 is induced by hypoxia and could be a protective factor against ischemic damage.

Figure 1. Anoxia increases NFAT5 protein abundance and promotes nuclear translocation.

A. 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. (*vs. 300 mosmol/normoxia and & vs. 640 mosmol/normoxia). B. NFAT5 cellular distribution after 2 hrs of anoxia evaluated in primary IMCD cells by immunofluorescence. Green = NFAT5 labelling (Alexa488); blue = nuclei (Hoechst 33258). C. 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). D. 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).

Figure 2. Hypoxia induces NFAT5 in cell culture.

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.

Figure 3. NFAT5 and HIF-1α are independently up-regulated by hypoxia.

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). A. Protein abundance of NFAT5 and HIF-1α were studied by Western blot in cells transfected with siRNA against NFAT5. 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.

Figure 4. NFAT5-regulators protein, ATM and PI3K, were induced in HEK293 cells by hypoxia.

HEK293 cells cultured at 300 mosmol were exposed for 0, 4, 8 and 16 hrs to hypoxia (2.5% of PO₂). A. Time course response of ATM phosphorylation (normalized by total ATM) was measured by Western blot. B. Time course response of PI3K activation was measured by Western blot of HIF-1α protein abundance (normalized by tubulin: Tub). C. AKT-308 phosphorylation (normalized by total AKT) was measured by Western blot. A representative picture is shown in the upper section. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.

Figure 5. Wortmannin inhibited the NFAT5 activation by hypertonicity and hypoxia in HEK293 cells.

A. 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. 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. C. 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.

Figure 6. NFAT5 has a protective role against hypoxia.

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. A. Western blot of NFAT5. B. LDH activity was assayed in cell culture media and cell lysate by spectrometric determination of NADH. C. Western blot of M30. D. Western blot of Cleaved caspase-3. Bar graph represents Mean ± SEM. *, P<0.05; n = 5.

Figure 7. Kidney function after experimental I/R.

A. Tissue damage evaluated by PAS staining. Brush border, epithelial flattening and mitosis were present in kidneys from I/R animals (arrows). B. Serum creatinine (mg/dl) of sham and 24–96 hrs post-ischemia. C. 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.

Figure 8. NFAT5 and HIF-1α are induced in post ischemic kidneys.

A. Kidney sections of sham and I/R animals (72 hrs) were incubated with rabbit anti-NFAT5. Representative pictures of medulla from sham and I/R animals are shown. Preimmune serum did not stain significantly (data not shown). Scale bar = 100 µm. The arrows indicate the localization of the corresponding marker for NFAT5. B. NFAT5 mRNA abundance in cortex and medulla of kidneys were determined by qRT-PCR. C. NFAT5 protein abundance in cortex and medulla of kidneys were determined by Western blot D. HIF-1α (protein abundance in cortex and medulla of kidneys was determined by Western blot. A representative picture is shown in the upper section. Bar graph represents Mean ± SEM. * or & indicates P<0.05; n = 5 (* vs sham medulla and & vs sham cortex).

Figure 9. Experimental I/R induced renal Aldose Reductase (AR) expression.

A. 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. AR mRNA abundance in kidney cortex and medulla measured by qRT-PCR. Bar graph represents Mean ± SEM. * or & indicates P<0.05; n = 5 (*vs sham medulla & vs sham cortex).

Figure 10. NFAT5-regulators protein, ATM and PI3K, were induced in post-ischemic kidneys.

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. Relative ATM abundance. C. Relative p110α abundance. Bar graph represents Mean ± SEM. * or & indicates P<0.05; n = 5 (* vs sham medulla & vs sham cortex).