Electrolyte Blood Press.  2010 Dec;8(2):59-65.

Membrane Trafficking of Collecting Duct Water Channel Protein AQP2 Regulated by Akt/AS160

• Jung HJ
• Kwon TH

Abstract

Akt (protein kinase B (PKB)) is a serine/threonine kinase that acts in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. The PI3K/Akt signaling pathway, triggered by growth factors and hormones including vasopressin, is an important pathway that is widely involved in cellular mechanisms regulating transcription, translation, cell growth and death, cell proliferation, migration, and cell cycles. In particular, Akt and Akt substrate protein of 160 kDa (AS160) are likely to participate in the trafficking of aquaporin-2 (AQP2) in the kidney collecting duct. In this study, we demonstrated that 1) small interfering RNA (siRNA)-mediated gene silencing of Akt1 significantly decreased Akt1 and phospho-AS160 protein expression; and 2) confocal laser scanning microscopy of AQP2 in mouse cortical collecting duct cells (M-1 cells) revealed AS160 knockdown by siRNA increased AQP2 expression in the plasma membrane compared with controls, despite the absence of dDAVP stimulation. Thus, the results suggest that PI3K/Akt pathways could play a role in AQP2 trafficking via the AS160 protein.

Keyword

aquaporin 2; protein kinase B; protein transport; rab GTP-binding proteins; TBC1D4 protein

MeSH Terms

Animals
Aquaporin 2
Cell Cycle
Cell Death
Cell Membrane
Deamino Arginine Vasopressin
Gene Silencing
Intercellular Signaling Peptides and Proteins
Kidney Tubules, Collecting
Membranes
Mice
Microscopy, Confocal
Phosphatidylinositol 3-Kinase
Phosphotransferases
Protein Transport
Proto-Oncogene Proteins c-akt
rab GTP-Binding Proteins
RNA, Small Interfering
Vasopressins
Water
Aquaporin 2
Deamino Arginine Vasopressin
Intercellular Signaling Peptides and Proteins
Phosphatidylinositol 3-Kinase
Phosphotransferases
Proto-Oncogene Proteins c-akt
RNA, Small Interfering
Vasopressins
Water
rab GTP-Binding Proteins

Figure

• 

  Fig. 1 Regulation of aquaporin-2 (AQP2) in response to dDAVP treatment in mouse cortical collecting duct cells (M-1 cells). A and B) Immunofluorescence microscopy of AQP2. Short-term dDAVP-treatment (10-8 M) for 15 minutes in M-1 cells increased AQP2 trafficking to the plasma membrane (indicated by arrows in B), compared with vehicle-treated M-1 cells (A). C and D) Semiquantitative immunoblotting of AQP2. Immunoblots reacted with anti-AQP2 revealed 29- and 35- to 50-kDa AQP2 bands, representing nonglycosylated and glycosylated forms of AQP2, respectively. AQP2 expression was significantly increased in 2 day-dDAVP-treated M-1 cells, compared with either vehicle-treated or 1 day-dDAVP-treated M-1 cells. n, the number of separate M-1 cell lysates.*P < 0.05 when compared to the vehicle-treated control group.†P < 0.05 when compared to the 1 day-dDAVP treated group.

• 

  Fig. 2 Semiquantitative immunoblotting of Akt1 and AQP2, aquaporin-2 (AQP2) in mouse cortical collecting duct cells (M-1 cells) with Akt1 knockdown. A and B) Immunoblots reacted with anti-Akt1 revealed 60-kDa Akt1 bands. Akt1-directed small interfering RNA (siRNA) treatment decreased Akt1 expression. C and D) Immunoblots reacted with anti-AQP2 revealed 29- and 35- to 50-kDa AQP2 bands, representing nonglycosylated and glycosylated forms of AQP2, respectively. AQP2 expression was unchanged in M-1 cells with Akt1 knockdown. n, the number of separate M-1 cell lysates.*P < 0.05 when compared to control non-targeting siRNA-transfected M-1 cells.

• 

  Fig. 3 Semiquantitative immunoblotting of Akt1, AS160, and p-AS160 in mouse cortical collecting duct cells (M-1 cells) with Akt1 knockdown. A-C) Akt1 knockdown was associated with unchanged or marginally increased total AS160 expression. In contrast, phospho-AS160 level normalized to the total AS160 protein values were markedly decreased (~160 kDa band on PAS immunoblots indicated by an arrow in panel A). siRNA, small interfering RNA; n, the number of separate M-1 cell lysates.*P < 0.05 when compared with the vehicle-treated control group (non-targeting siRNA-transfected M-1 cells).†P < 0.05 when compared with dDAVP-treated control group (non-targeting siRNA-transfected M-1 cells).

• 

  Fig. 4 Semiquantitative immunocytochemistry of aquaporin-2 (AQP2). AQP2-labeled mouse cortical collecting duct cells (M-1 cells) were randomly selected (asterisk in A) and total pixel intensity of the plasma membrane (PM) (C) and whole cell (both PM and cytosol except nucleus, D) were acquired respectively from the selected cells (B). E and F) AQP2 labeling in M-1 cells transfected with Con-small interfering RNA (Con-siRNA) or AS160-siRNA. G) The ratio of total pixel intensity in the PM to that of the whole cell (ratio of PM/Total) was calculated from randomly selected M-1 cells and averaged in each group. N, nucleus; n = number of cells randomly chosen and examined in each group.*P < 0.05 when compared to Con-siRNA. Bar, 10 µm.

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