EXPRESSION OF SYNAPTOTAGMIN-VIII IN RAT-KIDNEY

The synaptotagmins are a family of integral membrane proteins proposed to function as regulators of both exocytosis and endocytosis. Here, w e have used immunochemical techniques and RT-PCR to assess sites of re nal expression of synaptotagmin VIII. A polyclonal antibody was raised to a synthetic peptide corresponding to the carboxy-terminal 21 amino acids of mouse synaptotagmin VIII. On immunoblots of membrane fractio ns from renal cortex and medulla (and in several other tissues), the a ntibody labeled a 52-kDa band (absent with preimmune IgG). Immunofluor escence localization was carried out in tissue sections from rat kidne y. The synaptotagmin VIII antibody labeled early proximal tubules, thi n ascending limbs, thick ascending limbs, connecting tubules, and coll ecting ducts. In collecting ducts, both type A and B intercalated cell s exhibited basolateral labeling, whereas principal cells were labeled chiefly in the apical and subapical portion of the cells. Thick ascen ding limbs were labeled in both the basolateral and apical regions. RT -PCR experiments using total RNA extracted from cortex and medulla or microdissected inner medullary collecting ducts gave a single band of appropriate size. Sequencing of the PCR product confirmed that the amp lified target is synaptotagmin VIII. We conclude that synaptotagmin VI II is broadly expressed among renal tubule epithelia, raising the poss ibility that it is involved in regulation of transport and/or cell rem odeling at several sites in the nephron and collecting duct.