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 develo ped a ClC-5-specific rat monoclonal antibody. Immunoblotting demonstrated a n 85-kDa band of ClC-5 in the kidney and ClC-5 transfected cells. Immunocyt ochemistry revealed significant labeling of ClC-5 in brush-border membrane and subapical intracellular vesicles of the proximal tubule. In addition, a pical and cytoplasmic staining was observed in the type A intercalated cell s in the cortical collecting duct. In contrast, the staining was minimal in the outer and inner medullary collecting ducts and the thick ascending lim b. Western blotting of vesicles immunoisolated by the ClC-5 antibody showed the presence of H+-ATPase, strongly indicating that these two proteins wer e present in the same membranes. Double labeling with antibodies against Cl C-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 th at ClC-5 might be involved in the endocytosis and/or the H+ secretion in th e proximal tubule cells and the cortical collecting duct type A intercalate d cells in mouse kidney.