Peroxisome proliferator-activated receptor-γ agonists repress epithelial sodium channel expression in the kidney

Abstract

Thiazolidinediones (TZDs), known as peroxisome proliferator-activated receptor (PPAR) agonists, are used to treat type 2 diabetes. However, ∼5% of patients experience the treatment-limiting side effect of edema. Studies have implicated activation of the epithelial sodium channel (ENaC) as a cause of TZD-induced fluid retention, although there have been conflicting reports. The goal of this study was to resolve the role of PPARγ in control of ENaC isoforms in the kidney. Herein, we demonstrate in mice that rosiglitazone (RGZ), a PPARγ ligand, increases body weight and abdominal fat pad fluid content and reduces hematocrit. Seven days of RGZ decreases ENaCα and ENaCβ mRNA and ENaCγ protein expression in the kidney cortex, and acute treatment for 5 h with pioglitazone, another potent TZD, does not increase renal ENaC isoform mRNA or protein expression. Pioglitazone also decreases ENaCα and ENaCγ mRNA expression in a cortical collecting duct cell line. As no direct transcriptional studies had been conducted, we examined the PPARγ-dependent regulation of ENaC. Pioglitazone represses ENaCγ promoter activity, and this repression is partially relieved by inhibition of protein synthesis. Chromatin immunoprecipitation assays revealed that repression is associated with a decrease in histone H4K5 acetylation at the proximal ENaCγ promoter. In summary, TZDs do not increase ENaC mRNA expression in the kidney, and in fact repress the ENaCγ promoter via an indirect transcriptional mechanism.

Fig. 1.

Rosiglitazone (RGZ) increases body weight and fluid retention in mice. A: mice fed RGZ showed a significant increase in body weight after 1 day of feeding. B: hematocrit was significantly lower. C: fluid content of abdominal fat was significantly greater in the RGZ-fed mice. D: these RGZ-induced changes occurred in the absence of significant differences in plasma aldosterone levels. *P < 0.05 vs. control diet.

Fig. 2.

RGZ decreases epithelial sodium channel (ENaC)α and ENaCβ mRNA expression in kidney cortex. A: real-time PCR analysis of renal cortical sections from mice fed RGZ for 7 days showed a significant decrease in ENaCα and ENaCβ mRNA expression compared with standard diet (control) mice. B and C: cortical expression of the 85-kDa subunit of ENaCγ was reduced in the RGZ-fed mice. *P < 0.05 vs. control.

Fig. 3.

RGZ does not significantly alter ENaC isoform mRNA expression in kidney medulla. A: real-time PCR analysis. B and C: Western blotting of renal medullary sections from mice fed RGZ for 7 days showed no significant alterations in ENaC isoform expression compared with standard diet (control) mice.

Fig. 4.

Acute pioglitazone treatment decreases urinary Na/K excretion without increasing ENaC expression. A: five-hour treatment (25 mg/kg body wt ip) demonstrated a trend for pioglitazone (pioglit) to lower urinary [Na] without an effect on [K] excretion. The ratio of urinary [Na] to [K] concentration was significantly lower in the group administered pioglitazone; *P < 0.05 vs. control. There was no significant increase in cortical ENaC or Sgk-1 expression in the pioglitazone-treated group (B). C and D: whole kidney protein expression of ENaC isoforms was not increased in the pioglitazone-fed mice, and there was a trend for a reduction in the 85-kDa subunit of ENaCγ (P = 0.056). Ponceau staining verified equivalent loading of protein in each lane.

Fig. 5.

Pioglitazone decreases ENaCα and ENaCγ expression in M1 cells. M1 cortical collecting duct cells were treated for 24 h with pioglitazone (pioglit) or control (DMSO). Real-time PCR analysis showed that ENaCα and ENaCγ mRNA expression significantly decreased, and Sgk-1 mRNA expression significantly increased with pioglitazone 10-μM treatment compared with the control. *P < 0.05 vs. control. The membrane protein expression of ENaCα and ENaCγ did not correlate directly with the mRNA findings.

Fig. 6.

M1 cells can support peroxisome proliferator-activated receptor (PPAR)γ-dependent transactivation. M1 cells were cotransfected with acyl-CoA oxidase (AOX)₃-TK-Luc [a PPAR response element reporter vector (PPRE)] (40) and PPARγ (pCMX-PPARγ) or the empty vector pCMX. In a dose-dependent manner, pioglitazone drove the PPRE reporter only when PPARγ was transfected into the system. *P < 0.05 vs. control (no PPARγ). **P < 0.05 vs. 10 nM pioglitazone + PPARγ.

Fig. 7.

Pioglitazone does not alter ENaCα promoter activity in M1 cells. M1 cells were transfected with an ENaCα luciferase reporter and PPARγ or the empty vector pCMX and then treated with varying concentrations of pioglitazone (pioglit). Pioglitazone did not activate or repress the ENaCα promoter construct.

Fig. 8.

ENaCγ promoter activity is repressed by PPARγ. M1 cells were transfected with ENaCγ promoter construct plasmids [containing 2926 (A) or 1248 (B) or 439 bp (C) of the proximal promoter] plus PPARγ or the empty vector pCMX, and they were treated with pioglitazone. In hENaCγ-p2926-Luc, there was a trend (P = 0.067) for PPARγ to repress ENaCγ promoter activity, independent of ligand addition (see also Fig. 9C). In all 3 reporter constructs, pioglitazone repressed ENaCγ promoter activity. D: representation of the proximal ENaCγ promoter showing multiple candidate PPREs. *P < 0.05 vs. control (no PPARγ). **P < 0.05 vs. control, + PPARγ.

Fig. 9.

PPARγ antagonist partially relieves pioglitazone-dependent repression of ENaCγ expression. M1 cells were pretreated with GW9662 (10 μM) or control (DMSO) for 30 min, followed by pioglitazone (pioglit) or control (DMSO) for 24 h. A: GW9662 is unable to relieve pioglitazone-dependent repression of ENaCα mRNA expression. *P < 0.05 vs. pioglit 0, GW9662 0. B: treatment with GW9662 relieved repression of ENaCγ mRNA expression. *P < 0.05 vs. pioglit 0, GW9662 0. NS, not significant. C: pioglitazone repressed activity of the hENaCγ-p2926 and D: hENaCγ-p439, but this effect was attenuated by GW9662 in hENaCγ-p439. *P < 0.05 vs. pioglit 0, GW9662 0, and pCMX.

Fig. 10.

Pioglitazone-induced repression of ENaCα and ENaCγ requires protein synthesis. M1 cells were treated with or without 75 ng/ml cycloheximide (CHX) and also with control (DMSO) or pioglitazone (10 μM). In the control group (no CHX) pioglitazone reduced ENaCα and ENaCγ mRNA expression. However, with CHX, there was a significant increase in ENaCα and ENaCγ, without any changes in ENaCβ expression. When pioglitazone was added to the CHX-treated cells, there was a trend for a reduction in ENaCα and ENaCγ expression compared with the CHX-treated control cells. *P < 0.05 vs. control (no CHX).

Fig. 11.

Repression of the ENaCγ promoter is associated with a decrease in histone H4K5 acetylation at the proximal promoter. M1 cells were treated with pioglitazone (5 μM) or control (DMSO) for 16 h and chromatin immunoprecipitation studies were performed using antibodies for Pol II, histone H4 acetlyation (H4), and histone H3 acetylation (H3) with anti-rabbit IgG as the negative control. Real-time PCR analysis was performed with primers for the proximal promoter and into the first 100 bp of transcription. *P < 0.05 vs. IgG control. **P < 0.05 vs. H4 control.