Abstract
Prolonged hypokalemia induces a decrease of urinary concentrating ability via down-regulation of aquaporin 2 (AQP2); however, the precise mechanisms remain unknown. To investigate the role of autophagy in the degradation of AQP2, we generated the principal cell-specific Atg7 deletion (Atg7Δpc) mice. In hypokalemic Atg7-floxed (Atg7f/f) mice, huge irregular shaped LC3-positive autophagic vacuoles accumulated mainly in inner medullary collecting duct (IMCD) cells. Total- and pS261-AQP2 were redistributed from apical and subapical domains into these vacuoles, which were not co-localized with RAB9. However, in the IMCD cells of hypokalemic Atg7Δpc mice, these canonical autophagic vacuoles were markedly reduced, whereas numerous small regular shaped LC3-negative/RAB9-positive non-canonical autophagic vacuoles were observed along with diffusely distributed total- and pS261-AQP2 in the cytoplasm. The immunoreactivity of pS256-AQP2 in the apical membrane of IMCD cells was markedly decreased, and no redistribution was observed in both hypokalemic Atg7f/f and Atg7Δpc mice. These findings suggest that AQP2 down regulation in hypokalemia was induced by reduced phosphorylation of AQP2, resulting in a reduction of apical plasma labeling of pS256-AQP2 and degradation of total- and pS261-AQP2 via an LC3/ATG7-dependent canonical autophagy pathway.
Highlights
Prolonged hypokalemia induces a decrease of urinary concentrating ability via down-regulation of aquaporin 2 (AQP2); the precise mechanisms remain unknown
Following 2 weeks of dietary K+ depletion, serum K+ and urinary K+-free diet (K+)excretion decreased on the K+-deficient diet in both Atg7flox/ flox mice (Atg7f/f) and Atg7Δpc mice compared with control groups
P62/sequestosome 1 (SQSTM1) was significantly increased only in the inner medullary collecting duct (IMCD) cells of Atg7Δpc mice especially with K+-depletion for 2 weeks (Fig. 1c,d), suggesting that, in the renal papilla, autophagy was www.nature.com/scientificreports selectively inhibited in the IMCD cells of Atg7Δpc mice. These findings suggest that the increased autophagic vacuoles in Atg7Δpc mice after K+-depletion represent the autophagy generated in an Atg7-independent manner (ATG5/ATG7-independent non-canonical autophagy)[36,37]
Summary
Prolonged hypokalemia induces a decrease of urinary concentrating ability via down-regulation of aquaporin 2 (AQP2); the precise mechanisms remain unknown. The immunoreactivity of pS256-AQP2 in the apical membrane of IMCD cells was markedly decreased, and no redistribution was observed in both hypokalemic Atg7f/f and Atg7Δpc mice These findings suggest that AQP2 down regulation in hypokalemia was induced by reduced phosphorylation of AQP2, resulting in a reduction of apical plasma labeling of pS256-AQP2 and degradation of total- and pS261-AQP2 via an LC3/ATG7-dependent canonical autophagy pathway. Www.nature.com/scientificreports the accumulation of cytoplasmic droplets in collecting duct cells[21,23,24] that are believed to represent lysosomal structures[20,25] and are labeled by AQP22 Even though these droplets are formed in a classical rodent model of hypokalemia induced by ingestion of K+ free diet for a period of 2 weeks, it is not certain whether they comprise autophagic vacuoles or are involved in AQP2 degradation. An ATG7/ATG5/LC3-II-independent pathway termed noncanonical (alternative) autophagy has recently been described[36,37,38,39,40,41]
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