Cellular and subcellular immunolocalization of ClC-5 channel in mouse kidney: Colocalization with H⁺-ATPase

To determine the immunolocalization of ClC-5 in the mouse kidney, we developed a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated an 85kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocytochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, apical and cytoplasmic staining was observed in the type A intercalated cells in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending limb. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of H⁺-ATPase, strongly indicating that these two proteins were present in the same membranes. Double labeling with antibodies against ClC-5 and H⁺-ATPase and analysis by confocal images showed that ClC-5 and H⁺-ATPase colocalized in these ClC- 5-positive cells. These findings suggest that ClC-5 might be involved in the endocytosis and/or the H⁺ secretion in the proximal tubule cells and the cortical collecting duct type A intercalated cells in mouse kidney.