Immunolocalization of aquaporin-8 in rat kidney, gastrointestinal tract, testis, and airways

The purpose of this study was to determine the cellular and subcellular loc alization of aquaporin-8 (AQP8) in rat kidney and other organs by RT-PCR an alyses and by immunoblotting and immunohistochemistry using peptide-derived rabbit antibodies to rat AQP8. RT-PCR and Southern blotting revealed the p resence of AQP8 mRNA in all kidney zones. LLC-PK1 cells transfected with a rat AQP8 construct exhibited strong labeling with the affinity-purified ant ibodies, whereas controls using cells transfected with the vector, but with out the insert, were negative. The labeling was almost exclusively associat ed with intracellular vesicles. Immunoblotting of kidney membrane fractions revealed a predominant single band of 26-28 kDa. AQP8 immunoreactivity was mainly present in the cortex and outer stripe of the outer medulla. Sequen tial ultracentrifugation of rat kidney membrane revealed that AQP8 resides predominantly in intracellular vesicular fractions. Immunocytochemistry rev ealed modest labeling of proximal tubules and weak labeling of collecting d ucts in cortex and medulla of rat kidney. The labeling was confined to cyto plasmic areas with no labeling of the brush border. Immunoblotting and RT-P CR/Southern blotting also revealed the presence of AQP8 protein and mRNA in rat liver, testis, epididymis, duodenum, jejunum, colon, and bronchi/trach ea. Consistent with this, immunohistochemistry revealed AQP8 labeling in th e hepatocytes and spematogenic cells in testis and in the basal cells in du ctus epididymis, trachea, and bronchial epithelia. Moreover, AQP8 labeling was observed in the myoepithelial cells in salivary, bronchial, and trachea l glands with no labeling of acini or ductal epithelial cells. AQP8 is also present in the surface epithelial cells in duodenum, jejunum, and colon. I n conclusion, AQP8 is expressed at low levels in rat kidney proximal tubule s and collecting ducts, and it is present in distinct cell types in liver, testis, epididymis, duodenum, jejunum, colon, trachea, and principal bronch i as well as in multiple glands, including salivary glands.