Albumin Stimulates Epithelial Na+ Transport and Barrier Integrity by Activating the PI3K/AKT/SGK1 Pathway

Abstract

Albumin is a major serum protein and is frequently used as a cell culture supplement. It is crucially involved in the regulation of osmotic pressure and distribution of fluid between different compartments. Alveolar epithelial Na⁺ transport drives alveolar fluid clearance (AFC), enabling air breathing. Whether or not albumin affects AFC and Na⁺ transport is yet unknown. We therefore determined the acute and chronic effects of albumin on Na⁺ transport in fetal distal lung epithelial (FDLE) cells and the involved kinase pathways. Chronic BSA treatment strongly increased epithelial Na⁺ transport and barrier integrity in Ussing chambers. BSA did not elevate mRNA expression of Na⁺ transporters in FDLE cells after 24 h. Moreover, acute BSA treatment for 45 min mimicked the chronic effects. The elevated Na⁺ transport was caused by an increased maximal ENaC activity, while Na,K-ATPase activity remained unchanged. Acute and chronic BSA treatment lowered membrane permeability, confirming the increased barrier integrity observed in Ussing chambers. Western blots demonstrated an increased phosphorylation of AKT and SGK1, and PI3K inhibition abolished the stimulating effect of BSA. BSA therefore enhanced epithelial Na⁺ transport and barrier integrity by activating the PI3K/AKT/SGK1 pathway.

Keywords: BSA; ENaC; Na+ transport; barrier integrity; lung; protein kinase B.

Figure 1

BSA increases epithelial Na⁺ transport after 24 h. FDLE cells were cultured in MEM and stimulated with BSA (1.0 g/L) for 24 h. Data are displayed as mean + SEM. (A) BSA significantly increased V_base, V_amil, and ΔV_amil (n = 55–56; ** p < 0.01; *** p < 0.001 by t-test). (B) BSA further significantly elevated R_te in FDLE cells (*** p < 0.001 by t-test).

Figure 2

Concentration-dependent effect of BSA. BSA increases epithelial Na⁺ transport after 24 h in a concentration-dependent manner. FDLE cells were cultured in MEM and stimulated with BSA (0.25 to 4.0 g/L) for 24 h. Data are displayed as mean + SEM. (A) BSA significantly increased ΔV_amil (n = 57–61; * p < 0.05; ** p < 0.01; *** p < 0.001 by one-way ANOVA with Dunnett’s post hoc test). (B) BSA further significantly elevated R_te in FDLE cells (* p < 0.05; ** p < 0.01 by one-way ANOVA with Dunnett’s post hoc test).

Figure 3

BSA does not affect ENaC mRNA expression. FDLE cells were cultured in MEM and stimulated with BSA (1.0 g/L) for 24 h. Data are displayed as mean + SEM normalized to control values (n = 8).

Figure 4

BSA increases epithelial Na⁺ transport after 45 min. FDLE cells were cultured in MEM and stimulated with BSA (1.0 g/L) for 45 min. Data are displayed as mean + SEM. BSA significantly increased (A) V_base, ΔV_amil, and (B) I_base (n = 67–69; * p < 0.05; ** p < 0.01; *** p < 0.001 by t-test). (C) BSA further significantly elevated R_te in FDLE cells after 45 min (** p < 0.01 by t-test).

Figure 5

BSA increases maximal ENaC activity after 45 min, but not that of the Na,K-ATPase. FDLE cells were cultured in MEM and stimulated with BSA (1.0 g/L) for 45 min. Data are displayed as mean + SEM. BSA significantly increased (A) amil_max and (B) R_te (n = 39–42; * p < 0.05; *** p < 0.001 by t-test). (C) In contrast, BSA had no effect on maximal Na,K-ATPase activity (ouab_max) after 45 min (n = 27–31).

Figure 6

BSA decreases membrane permeability. FDLE cells were cultured in MEM and stimulated with BSA (1.0 g/L) for 45 min and 24 h. Data are displayed as mean + SEM normalized to control values. BSA significantly decreased membrane permeability (n = 11–12; ** p < 0.01 by t-test).

Figure 7

BSA activates PI3K/AKT/SGK1 signaling pathway. FDLE cells were cultured in MEM and stimulated with BSA (1.0 g/L) for 45 min. Data are displayed as mean + SEM. BSA significantly increased (A) AKT and (B) NDRG1 phosphorylation (n = 11–13; * p < 0.05 by t-test). Western blots of pAKT and total AKT resulted in bands of 60 kDa. Western Blot of pNDRG1 and total NDRG1 resulted in bands of 46 and 48 kDa. (C) Western blot of pNEDD4L and total NEDD4L resulted in bands of 110 and 135 kDa (ns = non-significant).

Figure 8

PI3K inhibition prevents the BSA-induced increase in Na⁺ transport. FDLE cells were cultured in MEM, stimulated with BSA (1.0 g/L) and LY294002 (10 µM) for 24 h. Data are displayed as mean + SEM. (A) BSA significantly increased V_base, V_amil, and ΔV_amil, while LY294002 prevented the BSA-induced increase (n = 39–46; *** p < 0.001 by t-test; ns = non-significant). (B) BSA further significantly elevated R_te in FDLE cells, which was prevented by LY294002 (** p < 0.01 by t-test).

Figure 9

Transferrin has no effect on epithelial Na⁺ transport and R_te. FDLE cells were cultured in MEM and stimulated with transferrin (10 µg/mL) for 24 h. Data are displayed as mean + SEM. Transferrin had no effect on V_base, V_amil, ΔV_amil (A) or R_te (B) (n = 39–44).