Impaired AQP2 trafficking in Fxyd1 knockout mice: A role for FXYD1 in regulated vesicular transport

Abstract

The final adjustment of urine volume occurs in the inner medullary collecting duct (IMCD), chiefly mediated by the water channel aquaporin 2 (AQP2). With vasopressin stimulation, AQP2 accumulation in the apical plasma membrane of principal cells allows water reabsorption from the lumen. We report that FXYD1 (phospholemman), better known as a regulator of Na,K-ATPase, has a role in AQP2 trafficking. Daytime urine of Fxyd1 knockout mice was more dilute than WT despite similar serum vasopressin, but both genotypes could concentrate urine during water deprivation. FXYD1 was found in IMCD. In WT mice, phosphorylated FXYD1 was detected intracellularly, and vasopressin induced its dephosphorylation. We tested the hypothesis that the dilute urine in knockouts was caused by alteration of AQP2 trafficking. In WT mice at baseline, FXYD1 and AQP2 were not strongly co-localized, but elevation of vasopressin produced translocation of both FXYD1 and AQP2 to the apical plasma membrane. In kidney slices, baseline AQP2 distribution was more scattered in the Fxyd1 knockout than in WT. Apical recruitment of AQP2 occurred in vasopressin-treated Fxyd1 knockout slices, but upon vasopressin washout, there was more rapid reversal of apical AQP2 localization and more heterogeneous cytoplasmic distribution of AQP2. Notably, in sucrose gradients, AQP2 was present in a detergent-resistant membrane domain that had lower sedimentation density in the knockout than in WT, and vasopressin treatment normalized its density. We propose that FXYD1 plays a role in regulating AQP2 retention in apical membrane, and that this involves transfers between raft-like membrane domains in endosomes and plasma membranes.

FXYD protein isoforms differentially modulate human Na/K pump function

“…FXYD1 is known to be retained in the endoplasmic reticulum in some expression systems ( Lansbery et al, 2006 ), and coexpression of PKA increases its delivery to the plasma membrane ( Mounsey et al, 1999 ). More recently, FXYD1 was reported to regulate trafficking of aquaporins ( Arystarkhova et al, 2017 ). Inconsistencies in the literature regarding Na/K pump regulation by FXYD1 may relate to cell-specific issues, including the existence of phosphorylation-dependent FXYD1 pools that are not associated with Na/K pumps ( Wypijewski et al, 2013 ), but more probably highlight the fact that regulation of Na/K pump is very complex ( Lu et al, 2016 ) and may involve endocytosis events that are linked to oxidative stress and ischemia-reperfusion injury ( Fine et al, 2011 ; Hilgemann and Fine, 2011 ; Lariccia et al, 2011 ; Lin et al, 2013 ), which may vary depending on the contents of whole-cell patch pipettes or the cell isolation and cell culture procedures used.…”

“…FXYD4 (corticosteroid hormone-induced factor; Attali et al, 1995 ) was also found in kidney, where its expression increases in response to corticosteroid hormone. FXYD1 (phospholemman; Palmer et al, 1991 ; Moorman et al, 1992 ) was found to be an important PKA and PKC protein target in the heart and skeletal muscle, and was later reported in brain and kidney ( Arystarkhova et al, 2017 ; Feschenko et al, 2003 ). FXYD6 (phosphohippolin) and FXYD7 seem to be exclusive to the nervous system ( Béguin et al, 2002 ; Delprat et al, 2007 ; Kadowaki et al, 2004 ), while FXYD3 (Mat-8; Morrison et al, 1995 ) and FXYD5 (dysadherin or related to ion channel; Ino et al, 2002 ) are expressed in several epithelia and appear to be up-regulated in certain cancers.…”

Diagnostic value of urinary RBP, ALB and AQP2 in neonatal hydronephrosis and the relationship with expression of MCP-1 in the prenatal maternal peripheral blood

“…This is due to the fact that when hydronephrosis occurs in the neonates, the concentration of AQP2 which carries on the trans-membrane transportation of water is decreased, so that the water permeability of collecting ductis also decreased. As a result, the transmembrane transportation of water is hindered and the urine concentration is decreased (18). After effective treatment, the amount of AQP2 on the cell membrane is increased, leading to an increase in water reabsorption.…”

Mammalian urine concentration: a review of renal medullary architecture and membrane transporters

“…FXYD proteins are a family of small membrane proteins that regulate the transport properties of Na + -K + -ATPase, and several FXYD members are present in the kidney (Geering 2006). FXYD1 is expressed in the IMCDs of mice and dDAVP treatment induces dephosphorylation of FXYD1 and translocation of FXYD1 and AQP2 to the apical plasma membrane (Arystarkhova et al 2017). FXYD1 knockout results in dilute daytime urine with lower AQP2 abundance in the IM and reduced AQP2 retention in the apical membrane.…”

“…FXYD1 knockout results in dilute daytime urine with lower AQP2 abundance in the IM and reduced AQP2 retention in the apical membrane. Therefore, the role of FXYD1 may be to enhance AQP2 retention in the apical membrane of the IMCD (Arystarkhova et al 2017).…”