Abstract
The unique phenotype of renal medullary cells allows them to survive and functionally adapt to changes of interstitial osmolality/tonicity. We investigated the effects of acute hypertonic challenge on AQP2 (aquaporin-2) water channel trafficking. In the absence of vasopressin, hypertonicity alone induced rapid (<10 min) plasma membrane accumulation of AQP2 in rat kidney collecting duct principal cells in situ, and in several kidney epithelial lines. Confocal microscopy revealed that AQP2 also accumulated in the trans-Golgi network (TGN) following hypertonic challenge. AQP2 mutants that mimic the Ser(256)-phosphorylated and -nonphosphorylated state accumulated at the cell surface and TGN, respectively. Hypertonicity did not induce a change in cytosolic cAMP concentration, but inhibition of either calmodulin or cAMP-dependent protein kinase A activity blunted the hypertonicity-induced increase of AQP2 cell surface expression. Hypertonicity increased p38, ERK1/2, and JNK MAPK activity. Inhibiting MAPK activity abolished hypertonicity-induced accumulation of AQP2 at the cell surface but did not affect either vasopressin-dependent AQP2 trafficking or hypertonicity-induced AQP2 accumulation in the TGN. Finally, increased AQP2 cell surface expression induced by hypertonicity largely resulted from a reduction in endocytosis but not from an increase in exocytosis. These data indicate that acute hypertonicity profoundly alters AQP2 trafficking and that hypertonicity-induced AQP2 accumulation at the cell surface depends on MAP kinase activity. This may have important implications on adaptational processes governing transcellular water flux and/or cell survival under extreme conditions of hypertonicity.
Highlights
Most mammalian cells are exposed to an extracellular environment that remains isotonic under normal physiological conditions, largely as a result of renal regulatory mechanisms that maintain water and electrolyte body fluid composition within a very narrow range
In light of the key role that AQP2 plays in mediating luminal water entry into collecting duct (CD) principal cells, we investigated whether altered AQP2 trafficking and plasma membrane expression occur as a rapid response to hypertonic challenge
Following hypertonic challenge was not visibly altered by MAPK inhibition (Figs. 8B, 9B, and 10B, 1 and 3, and Figs. 8C, 9C, and 10C). These results reveal that simultaneous activation of p38, ERK1/2, and Jun N-terminal kinase (JNK) by hypertonicity is required for increased AQP2 expression at the cell surface but not the trans-Golgi region immediately following hypertonic challenge
Summary
Most mammalian cells are exposed to an extracellular environment that remains isotonic under normal physiological conditions, largely as a result of renal regulatory mechanisms that maintain water and electrolyte body fluid composition within a very narrow range. Immunofluorescence analysis of kidney slices (Fig. 1, G–I) further revealed that increased AQP2 expression at the plasma membrane in response to acute hypertonicity was mostly restricted to the apical side of principal cells of the proximal and distal inner medullary CD, whereas VP led to an accumulation of AQP2 at both apical and basal plasma membranes, as previously reported [20] (ratio of apical/basal AQP2 cell surface expression as revealed by image semiquantification: control, 0.89 Ϯ 0.09; NaCl, 3.83 Ϯ 0.33; VP, 1.69 Ϯ 0.21).
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