Hypotonicity Induces Aquaporin-2 Internalization and Cytosol-to-Membrane Translocation of ICln in Renal Cells

Abstract

Kidney collecting-duct cells swell in response to changes in medulla osmolality caused by the transition from antidiuresis to diuresis. Regulatory volume decrease (RVD) mechanisms must be activated to face this hypotonic stress. In Aquaporin-2 (AQP2)-expressing renal CD8 cells, hypotonicity decreased cell surface expression of AQP2 and increased the amount of AQP2 localized intracellularly, whereas the total amount of AQP2 phosphorylated at ser-256 decreased. Analysis of cAMP dynamics using fluorescence resonance energy transfer (FRET) showed that hypotonicity causes a reduction of cAMP, consistent with a decrease in phospho-AQP2. Moreover, hypotonicity caused a profound actin reorganization, associated with the loss of stress fibers and formation of F-actin patches (microspikes) at the cell border. Those changes were regulated by the monomeric GTPase Cdc42. Interestingly, expression of the dominant-negative Cdc42 (N17-Cdc42) prevented the hypotonicity-induced microspike formation and the generation of Cl(-) currents. Hypotonicity also caused the relocation from the cytosol to the plasma membrane and increase in interaction with actin of ICln (nucleotide-sensitive chloride current protein), which is essential for the generation of ion currents activated during RVD. Together, the profound actin remodeling, internalization of AQP2 and translocation of ICln to the plasma membrane during hypotonicity may contribute to RVD after cell swelling in renal medulla.

Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

“…Furthermore, it did not rescue the reduced cell surface expression of wild-type AQP2 in cells whose PKA activity was attenuated either by the PKA inhibitor PKI or following calmodulinsensitive adenylyl cyclase type 3 inhibition, which reduces intracellular cAMP. Previous observations showed decreased AQP2 phosphorylation in response to hypotonicity (21), indicating that extracellular tonicity modulates the phosphorylation state of AQP2 at Ser 256 . The observation that reduced AQP2 cell surface expression in LLC-AQP2 (S256D) cells treated with adenylyl cyclase type 3 inhibitors could be partially restored by hypertonicity, but not VP stimulation, suggests that in addition to AQP2 Ser 256 phosphorylation, other PKA-mediated phosphorylation events are required for AQP2 cell surface accumulation.…”

“…Renal medullary cells are routinely exposed to abrupt variations of extracellular tonicity that challenge cell volume homeostasis. Induction of AQP2 internalization from the surface of renal CD8 cells was recently shown to occur immediately following hypotonic challenge (21), possibly reflecting a role that AQP2 may play in regulatory volume decrease mechanisms. In light of the key role that AQP2 plays in mediating luminal water entry into CD principal cells, we investigated whether altered AQP2 trafficking and plasma membrane expression occur as a rapid response to hypertonic challenge.…”

Macromolecular crowding effect is critical for maintaining SIRT1's nuclear localization in cancer cells

“…Intriguingly, hypotonic stress has been shown to induce translocation of other proteins as well as activation of different cellular pathways. [43][44][45][46][47] It is currently unknown whether the SIRT1 leakage during cell fractionation is associated with any hypotonic stress response or has any physiological or pathological functions.…”

Glutathionylation of the Aquaporin-2 Water Channel

“…To immunoprecipitate and detect the total amount of AQP2, we used antibodies (Pre-C-tail Ab) against the 20-amino acid residue segment just N-terminal from the polyphosphorylated region of AQP2 (CLKGLEPDTDWEEREVRRRQ) (11). Alternatively, AQP2 was detected using a specific antibody (C-tail Ab) raised against a synthetic peptide corresponding to the last 15 C-terminal amino acids of human AQP2 (12). Glutathione antibodies (D8), NADPH, and lucigenin were purchased from Santa Cruz Biotechnologies (DBA, Italy).…”