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Am J Physiol Renal Physiol 281: F546-F556, 2001;
0363-6127/01 $5.00
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Vol. 281, Issue 3, F546-F556, September 2001

SNAP-25-associated Hrs-2 protein colocalizes with AQP2 in rat kidney collecting duct principal cells

Alok Shukla1, Henrik Hager2, Thomas Juhl Corydon3, Andrew J. Bean4, Ronald Dahl1, Zsolt Vajda2, Hui Li2, Hans Jürgen Hoffmann1, and Søren Nielsen2

1 Department of Respiratory Diseases, Aarhus University Hospital, 2 The Water and Salt Research Center, and 3 Institute of Human Genetics, University of Aarhus, DK-8000 Aarhus, Denmark; and 4 Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, Texas 77030

The vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct is likely mediated by vesicle-targeting proteins (N-ethylmaleimide-sensitive factor attachment protein receptors). Hrs-2 is an ATPase believed to have a modulatory role in regulated exocytosis. To examine whether Hrs-2 is expressed in rat kidney, we carried out RT-PCR combined with DNA sequence analysis and Northern blotting using a digoxigenin-labeled Hrs-2 RNA probe. RT-PCR and Northern blotting revealed that Hrs-2 mRNA is localized in all zones of rat kidney. The presence of Hrs-2 protein in rat kidney was confirmed by immunoblotting, revealing a 115-kDa protein in kidney and brain membrane fractions corresponding to the expected molecular size of Hrs-2. Immunostaining and confocal laser scanning microscopy of LLC-PK1 cells (a porcine proximal tubule cell line) transfected with Hrs-2 DNA confirmed the specificity of the antibody and revealed that Hrs-2 is mainly localized in intracellular compartments, including cathepsin D-containing lysosomal/endosomal compartments. The cellular and subcellular localization of Hrs-2 in rat kidney was examined by immunocytochemistry and confocal laser scanning microscopy. Hrs-2 immunoreactivity was observed in collecting duct principal cells, and weaker labeling was detected in other nephron segments. The labeling was predominantly present in intracellular vesicles, but labeling was also observed in the apical plasma membrane domains of some cells. Colabeling with AQP2 revealed colocalization in vesicles and apical plasma membrane domains, suggesting a role for Hrs-2 in regulated AQP2 trafficking.

vesicle-targeting receptors; N-ethylmaleimide-sensitive factor attachment protein receptors; collecting duct; aquaporin-2


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