Toshiaki Higashida1, Xun-Ting Zeng1, Mitsuyoshi
Hara1, Koichiro Omori2, Sei Sasaki3, Michiaki
Fujio Shimizu4 and Chiyoko Inagaki1,*
1Department╩of Pharmacology and 2Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
3Second╩Internal Medicine, Tokyo Medical and Dental University, Tokyo 113-8519, Japan
4Department╩of Cell Biology, Institute of Nephrology, Niigata University School of Medicine, Niigata 951-8510, Japan
*╩To whom correspondence should be addressed.
Abstract: In order to demonstrate the localization of an ethacrynic acid-sensitive Cl- pump in the rat kidney, immunohistochemical analysis was performed using an anti-Cl- pump antibody raised against rat brain Cl- pump protein with confocal laser scanning microscopy. The antibodies against Na+,K+-ATPase, aquaporin╩2 and a type╩B intercalated cell marker, 43-kDa protein, were also used for comparison. Anti-Cl- pump antibody recognized a 51-kDa renal protein of the same size as that in the brain on Western blots. Cl--pump-like immunoreactivity was observed on the basolateral membranes of 42╩▒╩3% of cortical collecting duct (CCD) cells and of 38╩▒╩1% of outer medullar collecting duct (OMCD) cells. Such immunoreactivity in CCD was sometimes co-localized with Na+,K+-ATPase, but in OMCD, the Cl- pump-like immunoreactivity co-existed with neither Na+,K+-ATPase, aquaporin╩2 nor the type╩B intercalated cell marker 43-kDa protein. Thus, the Cl- pump was demonstrated to be localized on the basolateral membranes of type╩A intercalated cells of cortical and medullary collecting ducts in the rat kidney.
Keywords: Cl- pump, Ethacrynic acid, Intercalated cell, Collecting duct, Kidney