The polarity of epithelial cells is critical for proper function. Maintenance of polarity requires sustained proper sorting of proteins and lipids to either apical or basolateral membranes using distinct sorting signals. Compared to basolateral sorting signals, apical signals are not well characterized and can be present within the lumenal, transmembrane, or cytosolic regions of the protein. N-glycosylation has been identified as one of the apical sorting signals. The sialomucin endolyn (CD164) is a transmembrane protein that contains an apical sorting signal (N-glycans) in the lumenal domain and a lysosomal/basolateral targeting signal (YXXØ motif) in its cytoplasmic tail. It cycles between the apical surface and lysosomes of renal epithelial cells and is expressed in embryonic and adult kidney. The first objective of this research was to dissect the specific determinant on N-glycosylation for endolyn apical sorting using a lipofectamine-mediated RNAi approach. The results demonstrated that sialylation but not branching of N-glycans is required for endolyn proper delivery. Futher, knockdown of galectin-9 (but not galectins 3, 4 or 8) selectively disrupted endolyn polarity suggesting that interaction between endolyn and galectin-9 is critical for endolyn apical delivery. Next, the function of endolyn in pronephric kidney was investigated during development using zebrafish as a model system. Knockdown of zebrafish endolyn using a translational inhibiting morpholino resulted in pericardial edema, hydrocephaly, and body curvature, suggesting a possible osmoregulation defect. Although the pronephric kidney appeared normal morphologically, clearance of fluorescent dextran was delayed, indicating an imbalance in water regulation in morphant embryos. Rescue experiments using rat endolyn mRNA revealed that both apical sorting and endocytic/lysosomal targeting are required for endolyn function during development of the zebrafish pronephric kidney. This work broadens our understanding of apical sorting mechanisms in polarized cells as well as its significance on kidney function and development.