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).

INTRODUCTION: The epithelial sodium channel (ENaC) 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; I/R group, in which both renal arteries were occluded for 30 min, and sham group. Data were collected 48 h post-I/R. To confirm AKI, 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 as a housekeeping 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: AKI 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. CONCLUSION: 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.