High-Salt Loading Downregulates Nrf2 Expression in a Sodium-Dependent Manner in Renal Collecting Duct Cells

Abstract

Background: High salt intake is associated with both oxidative stress and chronic kidney disease (CKD) progression. Nuclear factor E2-related factor 2 (Nrf2) is a transcriptional factor regulating the antioxidant and detoxifying genes to potently antagonize oxidative stress. This study examined the effect of high salt loading on the expression of Nrf2 in kidney.

Methods: Mice were treated with acute salt loading, and Nrf2 expression in the kidney was detected by Western blotting and immunostaining. Reactive oxygen species (ROS) levels in the kidney were measured using dihydroethidium (DHE) staining. In vitro, mpkCCD cells were cultured in high osmolality medium by adding sodium chloride (NaCl), sodium gluconate (Na-Glu), choline chloride (Choline-Cl), or mannitol. Then, Nrf2 and its target genes were measured.

Results: Nrf2 protein in renal cortex and medulla tissue lysates was significantly downregulated after acute salt loading. Immunofluorescence data showed that Nrf2 was mainly located in collecting duct principal cells evidenced by co-staining of Nrf2 with AQP2. Contrasting to the reduced Nrf2 expression, ROS levels in the kidney were significantly increased after salt loading. In vitro, the Nrf2 protein level was downregulated in mpkCCD cells after NaCl treatment for 24 h. Interestingly, sodium gluconate had a similar effect on downregulating Nrf2 expression as NaCl, whereas neither Choline-Cl nor mannitol changed Nrf2 expression. Meanwhile, the mRNA levels of Nrf2 target genes were downregulated by NaCl and/or sodium gluconate, while some of them were also regulated by Choline-Cl, indicating a more complex regulation of these genes under a high salt condition. Finally, we found that the downregulation of Nrf2 caused by NaCl was not affected by N-acetylcysteine (NAC), spironolactone, or NS-398, suggesting other mechanisms mediating Nrf2 downregulation caused by high salt challenge.

Conclusion: High salt downregulated Nrf2 mainly via a sodium-dependent manner in kidney collecting duct cells, which might contribute to the excessive renal oxidative stress and CKD progression.

Keywords: chronic kidney diseases; collecting duct cells; high salt; nuclear factor E2-related factor 2; oxidative stress.

FIGURE 1

Nrf2 protein expression in kidneys after acute high salt loading. (A) Western blotting analysis of Nrf2 and β-actin in renal cortex. (B) Densitometric analysis of Nrf2 normalized by β-actin in cortex tissue. (C) Western blotting analysis of Nrf2 and β-actin in renal medulla. (D) Densitometric analysis of Nrf2 normalized by β-actin in medulla tissue. The presented data are means ± SE. Control (CTR): n = 5; high salt loading (HS): n = 4.

FIGURE 2

Immunohistochemistry of Nrf2 in kidneys after acute high salt loading. (A) Immunohistochemistry of Nrf2 in renal cortex and medulla. Magnification: 400×. Scale bar: 20 μm. (B) Densitometric analysis of Nrf2 in renal cortex. (C) Densitometric analysis of Nrf2 in renal medulla. The presented data are means ± SE. The images shown are representative of four to five animals per group.

FIGURE 3

Co-localization of Nrf2 and AQP2 in normal kidneys. (A) Immunofluorescent co-labeling with anti-Nrf2 and anti-AQP2 antibody in normal kidneys. Nrf2 is shown as red color. AQP2 is shown as green color. DAPI staining is shown as blue color. Magnification: 400×. Scale bar: 20 μm. (B) Analysis of Nrf2 antibody specificity for immunostaining by replacing Nrf2 antibody with murine IgG. The images shown are representative of five normal animals.

FIGURE 4

Time course analysis of ROS generation and α-ENaC protein expression in kidneys after acute high salt loading. (A) ROS levels were examined with DHE in the kidney after salt loading for 12 and 24 h. (B) Quantification of DHE fluorescence intensity in the kidney after salt loading for 12 and 24 h. (C) Western blotting analysis of α-ENaC and β-actin in renal cortex after salt loading for 6, 12, and 24 h. (D) Densitometric analysis of α-ENaC normalized by β-actin in cortex tissue. (E) Western blotting analysis of α-ENaC and β-actin in renal medulla after salt loading for 6, 12, and 24 h. (F) Densitometric analysis of α-ENaC normalized by β-actin in medulla tissue. The presented data are means ± SE. n = 3–4 per group. ^∗p < 0.05 vs. CTR (control); ^#p < 0.01 vs. CTR (control).

FIGURE 5

Time course analysis of Nrf2 protein expression in mpkCCD cells after sodium chloride (NaCl) treatment. (A) Western blotting analysis of Nrf2 and β-actin in mpkCCD cells after NaCl treatment for 12, 24, and 36 h. (B) Densitometric analysis of Nrf2 normalized by β-actin in mpkCCD cells after NaCl treatment for 12, 24, and 36 h. The presented data are means ± SE. n = 3 per group. ^∗p < 0.05 vs. CTR (control); ^#p < 0.01 vs. CTR (control).

FIGURE 6

The protein and mRNA expression of Nrf2 in mpkCCD cells after the treatments of NaCl, Na-Glu, choline-Cl, and mannitol. (A) Western blotting analysis of Nrf2 and β-actin. (B) Densitometric analysis of Nrf2 normalized by β-actin. (C) qRT-PCR analysis of Nrf2in mpkCCD cells. The presented data are means ± SE. n = 3 per group. ^∗p < 0.05 vs. CTR (control); ^#p < 0.01 vs. CTR (control).

FIGURE 7

mRNA expression of Nrf2 target genes in mpkCCD cells after treatments of NaCl, Na-Glu, choline-Cl, and mannitol. (A) qRT-PCR analysis of AKR7a5. (B) qRT-PCR analysis of AKR1a4. (C) qRT-PCR analysis of HO-1. (D) qRT-PCR analysis of GSTa4. The presented data are means ± SE. n = 3 per group. ^∗p < 0.05 vs. CTR (control); ^#p < 0.01 vs. CTR (control).

FIGURE 8

mRNA expression of Nrf2 in mpkCCD cells after treatment of NaCl in combination with NAC, spironolactone, or NS-398 treatment. All groups were treated with NaCl except CTR (control) group. NAC: N-acetylcysteine. The Nrf2 mRNA expression was determined by qRT-PCR. The presented data are means ± SE. n = 3 per group. ^∗p < 0.05 vs. CTR (control).