LOCALIZATION AND REGULATION OF THE RAT RENAL NA-K+-2CL(-) COTRANSPORTER, BSC-1()

To investigate the role of the thick ascending limb (TAL) Na+-K+-2Cl(- ) cotransporter in regulation of water excretion, we have prepared a p eptide-derived polyclonal antibody based on the cloned cDNA sequence o f the rat type 1 bumetanide-sensitive cotransporter, BSC-1 (also terme d ''NKCC-2''). Immunoblots revealed a single broad 161-kDa band in mem brane fractions of rat renal outer medulla and cortex but not from rat colon or parotid gland. A similar protein was labeled in mouse kidney . Immunoperoxidase immunohistochemistry in rat kidney revealed labelin g restricted to the medullary and cortical TAL segments. Because long- term regulation of urinary concentrating ability may depend on regulat ion of Na+-K+-2Cl(-) cotransporter abundance, we used immunoblotting t o evaluate the effects of several in vivo factors on expression levels of BSC-1 protein in rat kidney outer medulla. Chronic oral saline loa ding with 0.16 M NaCl markedly increased BSC-1 abundance. However, lon g-term vasopressin infusion or thirsting of rats did not affect BSC-1 abundance. Chronic furosemide infusion caused a 9-kDa upward shift in apparent molecular mass and an apparent increase in expression level. These results support the previous identification of BSC-1 as the TAL Na+-K+-2Cl- transporter and demonstrate that the expression of this tr ansporter is regulated.