Differential expression of AQP1 in microdomain-enriched membranes of renal cell carcinoma

Human aquaporin-1 (AQP1) is the most studied member of the aquaporin family, acting as molecular water channel. It is also considered a differentiation marker for proximal renal tubular cells, from which clear cells renal cell carcinoma (RCC) originates, playing an important role in urine formation. We therefore studied AQP1 expression at the proteomic level in RCC and normal tissues, mainly focusing on microdomain-enriched membranes in which AQP1 is highly concentrated. Subcellular fractions were prepared through differential centrifugation, and microdomain-enriched fractions were purified from a plasma membrane-enriched fraction by 1% Triton X-100 treatment followed by ultracentrifugation in sucrose gradient. After SDS-PAGE and Western blot analyses with antibodies against AQP1, lower expression levels of AQP1 isoforms were observed in each subcellular fraction of RCC compared to fractions from normal kidney tissues. The presence of AQP1 in the immunoreactive bands was verified by MALDI-TOF-MS and LC-ESI-MS/MS analysis. Glycosylation of AQP1 was also investigated using N-glycosidase F, confirming the presence of a N-glycosylated isoform of AQP1 in the 35-45-kDa region. These results highlight an under-expression of AQP1 protein and its glycosylated isoforms in homogenate and subcellular fraction obtained from RCC tissue compared to adjacent normal cortex.