Aquaporin-6: An intracellular vesicle water channel protein in renal epithelia

Abstract

All characterized mammalian aquaporins (AQPs) are localized to plasma membranes where they function chiefly to mediate water transport across cells. Here we show that AQP6 is localized exclusively in intracellular membranes in renal epithelia. By using a polyclonal antibody to the C terminus of AQP6, immunoblots revealed a major 30-kDa band in membranes from rat renal cortex and medulla. Endoglycosidase treatment demonstrated presence of an intracellular high mannose glycan on each subunit. Sequential ultracentrifugation of rat kidney homogenates confirmed that AQP6 resides predominantly in vesicular fractions, and immunohistochemical and immunoelectron microscopic studies confirmed that >98% of AQP6 is located in intracellular membrane vesicles. In glomeruli, AQP6 is present in membrane vesicles within podocyte cell bodies and foot processes. In proximal tubules, AQP6 is also abundant in membrane vesicles within the subapical compartment of segment 2 and segment 3 cells, but was not detected in the brush border or basolateral membranes. In collecting duct, AQP6 resides in intracellular membrane vesicles in apical, mid, and basolateral cytoplasm of type A intercalated cells, but was not observed in the plasma membrane. Unlike other members of the AQP family, the unique distribution in intracellular membrane vesicles in multiple types of renal epithelia indicates that AQP6 is not simply involved in transcellular fluid absorption. Moreover, our studies predict that AQP6 participates in distinct physiological functions such as glomerular filtration, tubular endocytosis, and acid-base metabolism.

Figure 1

Immunoblot analyses of tissue expression and N-glycosylation of AQP6. (A, Left) Anti-AQP6 immunoblot of membranes isolated from X. laevis oocytes injected with AQP6 cRNA or indicated rat kidney tissues (see Materials and Methods). (Right) Anti-AQP6 immunoblot of membrane proteins from outer medulla before (−) or after (+) digestion with peptide/N-glycosidase F (PNGase F) or endoglycosidase Hf (Endo H). (B) Immunoblots of kidney membrane fractions; pellets from sequential centrifugation at 1,000 ×, 4,000 ×, 17,000 ×, and 200,000 × g.

Figure 2

Immunocytochemical analyses of cellular and subcellular localization of AQP6 in kidney using immunoperoxidase labeling of cryostat (A, C, and E) and semithin cryosections (B, D, and F–H). (A) AQP6 labeling of straight proximal tubules (P) in medullary rays (MR, stippled lines) and glomeruli (G) in cortex (COR). (B) AQP6 in subapical domains of proximal tubule cells (arrows) but not in brush border (arrowheads) or basolateral plasma membranes. A sharp transition separates labeled from unlabeled proximal tubule cells (∗). AQP6 labeling of podocyte cell bodies (P) and foot processes (FP) but not of Bowman capsule (BC) or mesangial cells (M) of glomerulus. (C) Abundant AQP6 labeling of collecting duct in the outer medulla (OSOM, outer stripe; ISOM, inner stripe). (D) Abundant AQP6 labeling within type A intercalated cells (arrows). Absent AQP6 labeling in principal cells (arrowheads), thick ascending limbs (T), thin descending limbs, and vascular structures. (E) AQP6 within intercalated cells of collecting ducts in the inner stripe of outer medulla and inner medulla (IM). (F) AQP6 labeling of cytoplasm in type A intercalated cells (arrows) of inner medullary collecting duct. (G) Peptide-absorbed anti-AQP6 immunolabeling control of proximal tubule cells and (H) collecting duct cells in inner stripe of outer medulla. Magnifications: ×150 (A, C, and E); ×1,100 (B, D, and F–H).

Figure 3

Immunoelectron microscopy of AQP6 in ultrathin cryosections of glomerulus. Immunogold AQP6 labeling of intracellular structures (arrowheads) in foot processes and cell bodies of podocytes; no labeling of endothelial and mesangial cells (A and B). Some AQP6 labeling associated with larger bodies resembling lysosomes or multivesicular bodies (∗, C). BM, basement membrane; E, endothelial cell; FP, foot process; PC, podocyte cell body. Magnification: ×60,000.

Figure 4

Immunoelectron microscopy of AQP6 in segment 3 proximal tubule cell. AQP6 labeling of small vesicles (arrowheads) and dense apical tubules in the subapical part of the cell. BB, brush border; M, mitochondria; V, vesicle. Magnification: ×85,000.

Figure 5

Immunoelectron microscopy of AQP6 in collecting duct type A intercalated cell from inner stripe of outer medulla. AQP6 labeling of small vesicles and tubulocisternal profiles (arrowheads). (Inset) Ultrathin cryosection of apical part of type A intercalated cell. AQP6 labeling of vesicles and tubulocisternal profiles (arrowheads). MP, microplicae; M, mitochondria; N, nucleus; T, tubulocisternal profiles; V, vesicles. Magnification: ×60,000.