Increased renal ENaC subunits and sodium retention in rats with chronic heart failure

Abstract

Renal tubular dysfunction could be involved in the increased sodium and water reabsorption in chronic heart failure (CHF). The goal of the present study was to examine the molecular basis for the increased renal sodium and water retention in CHF. We hypothesized that dysregulation of renal epithelial sodium channels (ENaC) could be involved in the pathogenesis of CHF. The left coronary ligation-induced model of heart failure in the rat was used. Real-time PCR and Western blot analysis indicated that the mRNA and protein abundance of α-, β-, and γ-subunits of ENaC were significantly increased by in the cortex (mRNA: α-ENaC Δ104 ± 24%, β-ENaC Δ47 ± 16%, γ-ENaC Δ55 ± 18%; protein: α-ENaC Δ114 ± 28%, β-ENaC Δ150 ± 31%, γ-ENaC Δ39 ± 5% compared with sham rats) and outer medulla (mRNA: α-ENaC Δ52 ± 18%, β-ENaC Δ38 ± 8%, γ-ENaC Δ39 ± 13%; protein: α-ENaC Δ88 ± 16%, β-ENaC Δ94 ± 28%, γ-ENaC Δ45 ± 9% compared with sham rats) of CHF compared with sham-operated rats. Immunohistochemistry microscopy confirmed the increased labeling of α-, β-, and γ-ENaC subunits in the collecting duct segments in rats with CHF. Furthermore, there was a significant increase in diuretic (7-fold compared with sham) and natriuretic responses (3-fold compared with sham) to ENaC inhibitor benzamil in the rats with CHF. Absence of renal nerves produced a greater contribution of ENaC in sodium retention in rats with CHF. These results suggest that the increased expression of renal ENaC subunits may contribute to the renal sodium and water retention observed during CHF.

Fig. 1.

A: relative mRNA expression of epithelial sodium channel (ENaC) α-, β-, and γ-subunits in the cortex of sham and chronic heart failure (CHF) rats as measured by real-time PCR. B: relative mRNA expression of ENaC subunits in the outer medulla of sham and CHF rats as measured by real-time PCR. *P < 0.05 compared with respective sham group.

Fig. 2.

Immunoblotting for ENaC in the cortex of sham and CHF rats. A: an image of the bands detected from 40 to 220 kDa using α-, β-, and γ-ENaC antibodies in sham (first lane) and CHF rats (second lane). B: example of visualized electrophoresis bands of ENaC subunits and GAPDH in sham and CHF rats. C: mean data for band densities of ENaC subunits normalized by GAPDH in sham and CHF rats. *P < 0.05 compared with respective sham group.

Fig. 3.

Immunoblotting for ENaC in the outer medulla of sham and CHF rats. A: example of visualized electrophoresis bands of ENaC subunits and GAPDH in sham and CHF rats. B: mean data for band densities of ENaC subunits normalized by GAPDH in sham and CHF rats. *P < 0.05 compared with respective sham group.

Fig. 4.

Immunofluorescent staining of renal sections for α-ENaC in cortex (A) and medulla (B) of a sham and a CHF rat. An increase in immunostaining is noted in the CHF rat (magnification, ×400).

Fig. 5.

Immunofluorescent staining of renal sections for β-ENaC in cortex (A) and medulla (B) of a sham and a CHF rat. An increase in immunostaining is noted in CHF rat (magnification, ×400).

Fig. 6.

Immunofluorescent staining of renal sections for γ-ENaC in cortex (A) and outer medulla (B) of a sham and a CHF rat. An increase in immunostaining is noted in CHF rat (magnification, ×400).

Fig. 7.

A: urine flow in response to benzamil injection in sham and CHF rats with intact renal nerves. B: urine flow in response to benzamil injection in sham and CHF rats with denervated kidneys. C: urine flow changes in response to benzamil injection. *P < 0.05 compared with respective sham group.

Fig. 8.

A: sodium excretion in response to benzamil injection in sham and CHF rats with intact renal nerves. B: sodium excretion in response to benzamil injection in sham and CHF rats with denervated kidneys. C: sodium excretion changes in response to benzamil injection. *P < 0.05 compared with respective sham group.