Fig 2: Scheme showing alveolar fluid clearance process in the lung epithelium under normal condition and during decompensated CHFSodium is actively transported from alveolar space to the lungs’ interstitium and blood vessels; achieved mainly by apical ENaC and basolateral Na+/K+ ATPase located to Alveolar Epithelial Cells type I (AECI) and ATII (AECII). This results in the formation of osmotic gradient, which drives transcellular and paracellular movement of water molecules. In decompensated CHF there is upregulation of ENaC in the apical side and downregulation of Na+/K+ ATPase in the basolateral side, which may contribute to the development of lung edema.

Fig 3: Effect of dual versus single RAS blockade on the expression of total and cleaved ENaC subunits in the non-clipped kidney of renovascular hypertensive rats. Samples isolated from the non-clipped kidney of renovascular-hypertensive rats treated with enalapril (2K-1C + E, 20 mg/kg/day), losartan (2K-1C + L, 30 mg/kg/day), enalapril plus losartan (2K-1C + E + L, 30 and 20 mg/kg/day, respectively), or vehicle (2K-1C) and vehicle-treated control rats (2K) containing equal amounts of membrane proteins (75 µg for a-ENaC, ß-ENaC, and ?-ENaC and 20 µg for actin) were subjected to SDS-PAGE, after which the proteins were transferred to a PVDF membrane and incubated with primary antibodies. Actin was used as a loading control. Representative immunoblots of electrophoresed renal membrane proteins and graphical representation of the relative expression of (A) Full-length (FL) a-ENaC, (B) cleaved a-ENaC, (C) ß-ENaC, (D) cleaved ?-ENaC. The values represent individual measurements and the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs. 2K; ##P < 0.01, ###P < 0.001, and ####P < 0.0001 vs. 2K-1C; &&&P < 0.001 and &&&&P < 0.0001 vs. 2K-1C + E; $$P < 0.01 and $$$$P < 0.0001 vs. 2K-1C + L.

Fig 4: ENaC protein and mRNA expression in PCK rats fed diets with varying sodium content. (a) Western blotting showing expression levels of a-ENaC in the renal cortex of the PCK rats fed a SD, NS and HS diets, and a summary graph with densitometry values (normalized to ß-actin expression for the same samples). Each lane on the blot is one animal. (b) mRNA expression for a-, ß-, and ?-ENaC in the renal cortex of the PCK rats fed a SD, NS and HS diets. (c) Western blotting showing expression levels of AQP2 in the renal cortex of the PCK rats fed a SD, NS and HS diets, and a summary graph with densitometry values (normalized to ß-actin expression for the same samples). (d) miRNAs found to be differentially expressed (p < .05) in the cortical tissues of the PCK rats fed a SD, NS and HS diets. *, p < .05, **p < .01. (e) Western blots demonstrating total ENaC subunit expression in cultured mCCD cells transfected with a control scrambled miR sequence or miR9a-5p, with summary densitometry graphs (normalized to ß-Actin and relative to control mCCD expression). Both ß- and ?-ENaC are significantly decreased in mCCD cells over expressing miR9a-5p. * p < .05. Values in (b) and (c) were compared using a one-way ANOVA with a Tukey post-hoc test. For (d), differential expression was determined by DESeq2 method [34]. False discovery rate within statistical analysis was controlled for using the Benjamini-Hochberg method, p value cutoff was 0.05. Graphs with whiskers show mean value in each group and SEM. In (e), following a Shapiro-Wilk normality test, control and experimental values were compared using an unpaired student t-test.

Fig 5: ENaC-like channels recorded in SR and ISS cell lines. (A) Representative current traces showing single ENaC-like channels’ activity in SR and ISS cell lines recoded at different membrane potentials using patch-clamp electrophysiology. (B) Channel prevalence in successful GigaOhm seals in SR and ISS cell lines without stimulation with trypsin. Representative current traces are shown at -30 mV. (C) IV curve demonstrating lower single conductance of ENaC-like channels in a SR cell line with Na+ as main cation in the pipette vs. Li+. (D) Cumulative IV curves for single ENaC-like channels recorded in SR and ISS cell lines. (E) Summary graphs demonstrating reversal potential and single channel conductance (SR line: Erev = 34.1 ± 1.5 mV, n = 4, and 10.5 ± 0.7 pS n = 8 vs. ISS line: Erev = 33.6 ± 5.5 mV, n = 6, and 10.3 ± 0.8 pS, n = 5, t-test).