Supplementary Material for: The effect of renal ischemia/reperfusion on the renal expression of epithelial sodium channels in rat: possible role of neural precursor cell-expressed developmentally down-regulated protein (Nedd4-2)

Aim The epithelial sodium channel expressed in renal collecting duct epithelia plays a key role in regulating sodium balance. ENaC is regulated by the ubiquitin ligase Nedd4-2, which binds to ⍺ENaC and causes its retrieval, under low energy conditions due to the activation of AMP activated protein kinase (AMPK). Acute kidney injury (AKI) after ischemia reperfusion (I/R) disrupts sodium balance. In this study, we examined the impact of acute renal I/R on renal Na+ handling, ENaC subunits expression in renal membranes and the potential role of AMPK-Nedd4-2 pathway in this process. Methods: Adult Sprague-Dawley rats were randomized into two groups; ischemia/reperfusion (I/R) group, in which both renal arteries were occluded for 30 minutes, and sham group. Data was collected forty-eight hours post I/R. To confirm acute kidney injury, tubular damage and serum creatinine levels were assessed. Serum Na+ concentration and urinary sodium excretion rate were measured. Renal gene expression of α, β and γ-ENaC subunits was studied using RT-PCR with hydroxymethylbilane synthase (HMBS) as a house-keeping gene. Protein expression of α, β and γ-ENaC in renal homogenates and membrane fractions and protein expression of Nedd4-2, phospho-Nedd4-2, AMPK, and p-AMPK in renal homogenates were assessed by Western blot. Results were expressed as mean ±SEM. Control groups were compared to I/R groups using student t test and p<0.05 was set as significant. Results: Acute kidney injury after I/R was confirmed by revealing a significant tubular cell damage and the rise in serum creatinine levels (1.66± 0.14 in I/R vs. 0.64±0.09 in sham group, p<0.001). Serum Na+ concentration and Na+ excretion rate were not altered after I/R. Gene expression of ENaC subunits was not affected by I/R, however protein expression of α-ENaC was decreased in renal membranes (0.03±0.003 in I/R vs. 0.05±0.01 in sham group, p<0.05), with no change in β or γ expression. There were increases in the expressions of Nedd4-2 (0.27±0.04 vs. 0.09±0.03, p<0.05), p-Nedd4-2 (0.15±0.04 vs. 0.04±0.01, p<0.05), AMPK (0.09±0.01 vs. 0.03±0.02, p<0.05) and p-AMPK (1.05±0.2 vs. 0.6±0, p<0.05) indicating the activation of AMPK-Nedd4-2 pathway. Conclusions Ischemia reperfusion caused renal injury, however, did not affect renal sodium handling. A decrease in membrane expression of α-ENaC was detected without a change in total α-ENaC suggesting ENaC trafficking into the cell. This occurred with the concomitant activation of AMPK-Nedd4-2 pathway, which is known to cause retrieval of ENaC and decrease its membrane expression.