Question:

The epithelial sodium channel (ENaC) marks the tightly regulated rate-limiting step of sodium reabsorption in the aldosterone-sensitive distal nephron (ASDN). There are contrasting reports regarding ENaC degradation, with evidence for lysosomal and proteasomal degradation found in overexpression systems [1], but solely proteasomal degradation in cells endogenously expressing ENaC [2]. Recently, we reported that inhibiting the proteasome with MG132 stimulated ENaC-mediated Na⁺ transport. Our aim was: i) to confirm the stimulatory effect of inhibiting the proteasome with the potent and specific compound bortezomib; ii) to investigate the effects of lysosomal inhibitors; iii) to explore the mechanism by which proteasomal inhibition stimulates ENaC-mediated Na⁺ transport.

Methods:

mCCD_cl1 cells were used as a murine model of principal collecting duct cells and were grown on permeable supports. ENaC-mediated Na⁺ transport was assessed by recording the equivalent short circuit current (I_SC) in Ussing chambers. Cell lysates were subject to western analysis to monitor phosphorylation of NDRG1, a specific downstream target of SGK1.

Results:

Apical application of bortezomib (1µM, 5h) stimulated I_SC by 2.9 ± 0.3 fold (n = 3). The stimulated I_SC was inhibited by amiloride, indicating that inhibition of proteasomal degradation stimulates ENaC-mediated Na⁺ transport. This result is consistent with our initital finding that 1µM MG132 stimulated I_SC by 2.8 ± 0.1 fold (n = 11). In contrast, the lysosomal inhibitors bafilomycin A (100nM), E64 (10nM), and leupeptin (5µM) did not alter I_SC when added to the apical bath. Interestingly, cells treated with the proteasomal inhibitor MG132 (n=3) displayed increased SGK1 activity as shown by a significant increase in NDRG1 phosphorylation. Moreover, the stimulation of I_SC due to MG132-treatment was significantly reduced when cells were pre-treated with the SGK1 inhibitor GSK650394 (n=4).

Conclusions:

Inhibition of proteasomal but not lysosomal degradation robustly stimulates ENaC-mediated Na⁺ transport in mCCD_cl1 cells. This stimulation appears to be dependent on SGK1 activity and may involve inhibition of channel retrieval from the plasma membrane. Our data indicate that inhibition of the proteasome indirectly stimulates ENaC by raising SGK1 activity rather than by preventing proteasomal ENaC degradation.

1. Staub, O. et al. (1997). EMBO Journal 16: 6325-36.

2. Malik, B. et al. (2005). Am J Physiol-Renal 289: F107-116.