Immunocytochemical studies of the na-k-cl cotransporter of shark kidney. Biemesderfer, Daniel, John Payne, Christian Lytle, and Bliss Forbush Iii. Departments of Internal Medicine and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven CT 06510 and Mount Desert Island Biological Laboratory, Salsbury Cove, Maine 04672
APStracts 3:0018F, 1996.
The Na-K-Cl cotransporter (NKCC or BSC) has been described in numerous secretory and reabsorptive epithelia and is an important part of the mechanism of NaCl reabsorption in both the mammalian and elasmobranch kidneys. We have recently developed a panel of four monoclonal antibodies (mAbs) raised to the 195 kDa Na-K-Cl cotransport protein of the shark rectal gland (sNKCC1) which is expressed along the basolateral plasma membrane of secretory cells in this tissue (29). Here, we report immunologic studies of the Na-K-Cl cotransporter in the kidney of the dogfish shark, Squalus acanthias. Western blot analysis of shark renal microsomes using mAbs J3, J7 and J25 identified proteins of approximately 195 and 150 kDa, while mAb J4 was not reactive. To define the cellular and subcellular distribution of the cotransport protein, immunofluorescence and immunoelectron microscopy studies were performed on fixed kidneys. Immunofluorescence microscopy on semithin (0.5 um) cryosections demonstrated that mAbs J3, J7 and J25 intensely stained the apical plasma membrane of all distal tubule segments. Weak staining was also seen along the basolateral membrane of most distal nephrons. Immunoelectron microscopy confirmed this observation and showed that some of these segments were morphologically similar to diluting segments from other species. mAbs also reacted with the brush border,and to a lesser extent the basolateral membrane, of proximal tubules. This study supports the hypothesis that the lateral bundle zone of the elasmobranch kidney functions as a countercurrent exchanger and is consistent with the presence of multiple isoforms of the Na-K-Cl cotransporter in the shark kidney. 

Received 2 March 1995; accepted in final form 13 December 1995.
APS Manuscript Number F71-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 29 January 96